SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF ANATOXIN-A(S)

by

Eric E. Buck

B.S., University of Minnesota, 2006

Submitted to the Graduate Faculty of

The Kenneth P. Dietrich School of Arts and Sciences

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

University of Pittsburgh

2012

ii

UNIVERSITY OF PITTSBURGH

The Kenneth P. Dietrich School of Arts and Sciences

This thesis was presented

by

Eric E. Buck

It was defended on

November 29th

, 2012

and approved by

Scott Nelson, Professor, Department of Chemistry

Kazunori Koide, Associate Professor, Department of Chemistry

Billy Day, Professor, Department of Pharmaceutical Science

Thesis Director: Peter Wipf, Distinguished University Professor, Department of Chemistry

iii

Copyright © by Eric E. Buck

2012

iv

Three routes toward the total synthesis of the neurotoxin anatoxin-a(s) have been explored. They

focus on the assembly of the backbone utilizing both Pd(0)- and Pd(II)-mediated processes. Our

current route leads to a late-stage precursor of anatoxin-a(s), hydroxy guanidine 134, in 18 steps

and in 8.0% overall yield utilizing an amidoalkylation step in the key cyclization.

SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF ANATOXIN-A(S)

Eric E. Buck, PhD

University of Pittsburgh, 2012

v

TABLE OF CONTENTS

1.0 SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF ANATOXIN-A(S) ......... 1

1.1 INTRODUCTION ................................................................................................................ 1

1.1.1 Cyanobacteria ................................................................................................... 1

1.1.2 Strategies for the formation of anatoxin-a(s)’s guanidine backbone:

Inspiration from related heterocycles. ....................................................................... 8

1.2 RESULTS AND DISCUSSION ......................................................................................... 20

1.2.1 Route 1: Palladium(0)-catalyzed diaminations ........................................... 21

1.2.2 Route 2: Palladium(II)-catalyzed amidocarbonylations ............................ 24

1.2.3 Route 3: Palladium(II)-catalyzed amidoalkylations ................................... 32

1.3 CONCLUSION ................................................................................................................... 50

2.0 EXPERIMENTAL ............................................................................................................. 51

3.0 REFERENCES ................................................................................................................... 90

APPENDIX A .............................................................................................................................. 94

vi

LIST OF TABLES

Table 1. Diamination of butadiene derivatives. ............................................................................ 18

Table 2. Conditions for the oxidation of silyl enol ether 72. ........................................................ 23

Table 3. Amidocarbonylation conditions. ..................................................................................... 26

Table 4. Conditions for attempted Curtius rearrangement of carboxylic acid 87. ........................ 30

Table 5. Conditions for attempted Hofmann rearrangement of amide 97. ................................... 32

Table 6. Screening conditions for key amidoalkylation of guanidine 99. .................................... 35

Table 7. Pd(II)-mediated cyclization of MOM-derivative 121. .................................................... 42

Table 8: Exploration of different bases to optimize phosphorylation........................................... 46

Table 9: Solvent screen under the phosphorylation conditions. ................................................... 47

Table 10: Exploring different phosphorylation conditions. .......................................................... 48

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LIST OF FIGURES

Figure 1. Structure of the cytotoxin aplysiatoxin (1). ..................................................................... 2

Figure 2. The structure of microcystin LR (2). ............................................................................... 3

Figure 3. The structure of nodularin (3). ......................................................................................... 3

Figure 4. Structure of cylindrospermopsin (4). ............................................................................... 4

Figure 5. Selected structures of neurotoxins produced by cyanobacteria. ...................................... 4

Figure 6: Structure identification of cyclization compounds and comparable urea compounds 81

and 82. ........................................................................................................................................... 27

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LIST OF SCHEMES

Scheme 1. Proposed mechanism of action of anatoxin-a(s). .......................................................... 6

Scheme 2. Biosynthesis of anatoxin-a(s) (8). ................................................................................. 7

Scheme 3: Synthesis of guanidine 15. ............................................................................................ 8

Scheme 4: Possible synthetic strategies for the synthesis of anatoxin-a(s). ................................... 9

Scheme 5: Two possible pathways for nucleopalladation. ............................................................. 9

Scheme 6. Synthesis of (+)-preussin. ............................................................................................ 10

Scheme 7. The synthesis of urea derivatives using the Pd2(dba)3/Xantphos catalyst system....... 11

Scheme 8: Intramolecular amination of alkenes through aminopalladation. ................................ 12

Scheme 9. Synthesis of (±)-alchorneine. ...................................................................................... 13

Scheme 10. Stereoselective formation of cyclic carbamates. ....................................................... 13

Scheme 11. Asymmetric synthesis of (-)-anatoxin-a. ................................................................... 14

Scheme 12. Synthesis of (+)-prosopinine. .................................................................................... 15

Scheme 13. Asymmetric intramolecular aminopalladation. ......................................................... 16

Scheme 14. Overman’s 2nd

generation catalysts. .......................................................................... 16

Scheme 15. Diamination of terminal olefins and proposed pathway. .......................................... 17

Scheme 16: Synthesis of bicyclic guanidine. ................................................................................ 18

Scheme 17. Shi's proposed mechanism for the diamination of olefins. ....................................... 19

Scheme 18. 1st Generation retrosynthetic analysis of anatoxin-a(s) (8). ...................................... 21

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Scheme 19. Attempted synthesis of silyl enol ether 68. ............................................................... 22

Scheme 20. Synthesis of TES silyl enol ether 72. ........................................................................ 23

Scheme 21. Ma's work on DMPU derivatives. ............................................................................. 24

Scheme 22. 2nd

Generation retrosynthetic analysis of anatoxin-a(s) (8). ..................................... 25

Scheme 23. Synthesis of allylic guanidine 78. ............................................................................. 25

Scheme 24. Proposed mechanism of amidocarbonylation. .......................................................... 28

Scheme 25. Synthesis of carboxylic acid 87 and proposed mechanism of ester isomerization. .. 29

Scheme 26. Radical decarboxylation of thiohydroxamate esters 94 and 95. ................................ 31

Scheme 27. Formation of amide 97. ............................................................................................. 31

Scheme 28. 3rd

Generation retrosynthetic analysis of anatoxin-a(s) (8). ...................................... 33

Scheme 29. Synthesis of thiourea 101. ......................................................................................... 33

Scheme 30. Synthesis of guanidine 99. ........................................................................................ 34

Scheme 31. Proposed catalytic cycle of amidoalkylation. ............................................................ 37

Scheme 32. Synthesis of alkyl azide 113. ..................................................................................... 38

Scheme 33. Synthesis of methyl carbamate 116. .......................................................................... 39

Scheme 34. Synthesis of N-hydroxy guanidine 9. ........................................................................ 40

Scheme 35. Improved synthesis of 99 and synthesis of MOM derivative 121............................. 41

Scheme 36: Trans-aminopalladation (AP) vs. cis-aminopalladation. .......................................... 43

Scheme 37. Synthesis of hydroxy guanidine 134. ........................................................................ 44

Scheme 38. A model system synthesis of 134 to explore phosphorylation conditions. ............... 45

Scheme 39: Attempted phosphorylation of 134............................................................................ 49

x

ACKNOWLEDGMENTS

I would like to first express my gratitude towards Dr. Wipf for providing me the opportunity to

work in his lab over the past several years. He has provided me with an environment that has

helped me develop into a better chemist, scientist, and person. I would be remiss if I did not

mention the wonderful molecule of the month competitions. The discussions and the freedom to

express my creativity during these events are some of my favorite memories of grad school. I

will never miss a β-leaving group no matter where my studies take me.

I would like to thank Dr. Nelson, Dr. Koide, and Dr. Day for being on my committee. I

have always enjoyed discussing not only my own chemistry during our various meetings but also

for their willingness to provide me with excellent feedback. I would like to give a special thanks

to Dr. Day for all the assistance he gave me on the BST3 LC-MS. That machine was invaluable

to my research. I am also very grateful to Dr. Floreancig. Even under the shortened timeline he

was always willing to discuss the chemistry and to provide extremely useful feedback. Thank

you.

I would like to take the time to acknowledge my fellow graduate students in the Wipf

group. I would like to thank Chenbo and Chad and later Nolan for providing some of the best

discussions as we worked on MOM. I could not thank Adam enough. Your role as a mentor and

friend cannot be adequately expressed in words. Last but not least I would like to thank Chris

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and Nate. For being there from day one. Kara you have my gratitude for willing to wait until

noon before badgering me.

Lastly, I need to thank my family for always being there. Thanks to my nephew who calls

me his friend Uncle Eric. You helped me more than you know little buddy.

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ABBREVIATIONS

Ac acetyl

AIBN 2,2’-azo bisisobutyronitrile

atm atmosphere

ATR Attenuated Total Reflectance

Boc tert-butoxycarbonyl

Bn benzyl

Bz benzoyl

Cbz benzyloxycarbonyl

DABCO 1,4-diazabicyclo[2.2.2]octane

dba dibenzylideneacetone

DCC dicyclohexylcarbodiimide

DCE 1,2-dichloroethane

DCM dichloromethane

DIAD diisopropyl azodicarboxylate

DIC diisopropyl carbodiimide

DIPA diisopropylamine

DIPEA diisopropylethylamine

DMAP N,N-4-dimethylaminopyridine

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DMF N,N-dimethylformamide

DMPU N,N-dimethyl propylene urea

DMSO dimethylsulfoxide

DPPA diphenylphosphoryl azide

dr diastereomeric ratio

DSS 4,4-dimethyl-4-silapentane-1-sulfonic acid

EDA ethylenediamine

EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ee enantiomeric excess

FDA Food and Drug Administration

Fmoc 9-fluorenylmethoxycarbonyl

HMPA hexamethylphosphoramide

IBBO (+)-2,2’-isopropylidenebis[(4R)-4-benzyl-2-

oxazoline)]

LD50 lethal dose required to kill 50% of the population

LDA lithium diisopropylamine

m-CPBA meta-chloroperbenzoic acid

MOM methoxymethyl

Ms methanesulfonyl

MVK methyl vinyl ketone

ND not determined

NMM N-methylmorpholine

NMP N-methylproline

xiv

NR no reaction

Ph phenyl

phthNH phthalimide

phthNK potassium phthalimide

PIDA phenyliodonium diacetate

PIFA phenyliodonium bis(trifluoroacetate)

PNS peripheral nervous system

Pyr pyridine

TBAB tetra-n-butylammonium bromide

TBAF tetra-n-butylammonium fluoride

TBDPS tert-butyldiphenylsilyl

TEA triethylamine

TES triethylsilyl

Tf trifluoromethanesulfonyl

TFA trifluoroacetic acid

TFAA trifluoroacetic anhydride

THF tetrahydrofuran

THP 2-tetrahydropyranyl

TMS trimethylsilyl

Ts p-toluenesulfonyl

1

1.0 SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF ANATOXIN-A(S)

1.1 INTRODUCTION

1.1.1 Cyanobacteria

Cyanobacteria, or blue-green algae, are prokaryotic microorganisms predominately found in

ponds, lakes, rivers and oceanic waters. Fossil records date these photosynthetic microorganisms

as far back as 2.15 billion years ago, and their ability to produce oxygen was a large factor in

turning earth’s early atmosphere into an oxygen-rich system.1 A rapid growth of cyanobacteria

can occur when large amounts of sunlight and food rich in nitrogen and phosphorus are present.

This rapid growth is referred to as an algal bacterial bloom and is often detrimental to

surrounding plant, animal, and human life.2,3

One example of a harmful algal bloom occurred in

1993 when a sudden increase in the salinity of several freshwater bodies of water in Florida, due

to a tidal surge, caused a dramatic decrease in local plant life.4 In the past 20 years,

cyanobacteria have emerged as interesting sources of biologically active secondary metabolites

that are commonly referred to as cyanotoxins. These cyanotoxins are classified into two groups

according to their modes of toxicity: cytotoxins that are assayed using cultured mammalian cells,

and highly lethal biotoxins that are assayed using small animals.3

2

While the cytotoxins are typically less deadly than the biotoxins, they are known for their

antialgal, antimycotic, and antibacterial properties. Cytotoxins also exhibit modest antitumor

activity.3 The presence of blue-green algae of the genus Lyngbya majuscula, which produces the

protein kinase C activator aplysiatoxin (1, Figure 1), on the surface of the red alga G.

coronopifolia was eventually determined to be the cause of food poisoning and dermatitis in

Hawaii in 1994 (Figure 1).4,5

Figure 1. Structure of the cytotoxin aplysiatoxin (1).

Biotoxins are highly lethal metabolites that are further divided into hepatotoxins and

neurotoxins. Hepatotoxins, or molecules that target the liver, include three common types:

microcystins, nodularins, and cylindrospermopsins. Microcystins are identified by their

macrocyclic heptapeptide backbone. The presence of five non-proteinogenic amino acids is

characteristic to microcystins, while the two proteinogenic amino acids are varied throughout the

microcystin family. Microcystin LR (microcystin leucine arginine) (2, Figure 2) is the most

lethal and abundant hepatotoxin with an LD50 of 25-125 g/Kg (mice). This natural product was

responsible for 50 fatal human poisonings at a hemodialysis center in Brazil.6 Its mechanism of

action consists of blocking protein phosphatases, leading to hemorrhaging of the liver.3

Another family of hepatotoxins, the nodularins, resembles the microcystins structurally

and has a characteristic pentapeptide macrocycle. These secondary metabolites are inhibitors of

3

serine/threonine protein phosphatase 1 and 2A, and this inhibition will lead to hemorrhaging.6

Nodularin (2, Figure 3), the parent compound in this family, possesses an LD50 of 50 – 70 g/Kg

(mice).7

Figure 2. The structure of microcystin LR (2).

Figure 3. The structure of nodularin (3).

Cylindrospermopsin (4, Figure 4), isolated from C. raciborskii, is a unique member of the

hepatotoxin family due to its lack of a macrocyclic peptide backbone. In 1979, it was

responsible for the hospitalization of 140 children on Palm Island, Queensland, Australia, and it

can also be found in Europe and America. Cylindrospermopsin exhibits an LD50 of 2.1 mg/Kg

(mice) and its mechanism of action is to block protein synthesis, leading to kidney and liver

failure. Recently, there is new evidence suggesting that protein synthesis inhibition is not the

only mechanism responsible for its toxicity.8,9

4

Figure 4. Structure of cylindrospermopsin (4).

In addition to producing cytotoxins, cyanobacteria produce several neurotoxins, in

particular anatoxin-a (5) and homoanatoxin-a (6), the saxitoxins (7), and the organophosphate

anatoxin-a(s) (8, Figure 5). Anatoxin-a, produced by several strains of Anabaena, is a neurotoxic

alkaloid with an LD50 of 200 – 250 g/Kg (mice). It binds to nicotinic receptors, leading to

respiratory failure within a few hours.2,6

As a result, 5 is commonly known as “very fast death

factor”. To date, there are several reported total syntheses of anatoxin-a and its structural analog

homoanatoxin-a, with the most recent being Stockman’s approach published in 2008.10

Homoanatoxin-a, isolated from Oscillatoria formosa, has a similar mechanism of action as

anatoxin-a.2

Figure 5. Selected structures of neurotoxins produced by cyanobacteria.

5

The saxitoxins, also known as paralytic shellfish poisons, block voltage gated sodium

channels, leading to neuromuscular paralysis and death by respiratory failure. With an LD50 of

10 g/Kg in mice, saxitoxin (7) is the most lethal of the saxitoxins and has been linked to a

number of sheep deaths in Australia.2,6

Anatoxin-a(s) (8) is an organophosphate isolated from Anabeana flos-aque in North

America, A. lemmermannii in Denmark, and Anabeana crassa in South America. In North

America, it has been linked to the deaths of dogs, birds, and swine; in Denmark it has been

associated with the death of wild birds.2,11,12

Anatoxin-a(s) was also detected in the Faxinal

Reservior (the main reservoir for the Caxias do Dul, Brazil), which commonly experiences

Anabeana crassa blooms. To date there are no reports of neurotoxicity caused by anatoxin-a(s)

in Brazil.13

It is a strong anticholinesterase (LD50 of 20 - 40 g/Kg in mice) that acts only in the

peripheral nervous system (PNS) and has a similar mechanism of action as the insecticide

paraoxon and the chemical weapon sarin.14-16

It is proposed that anatoxin-a(s) irreversibly

phosphorylates a serine residue in the active site of acetylcholinesterase. As shown in Scheme 1,

the hydroxyl functionality of an acetylcholinesterase serine residue adding into the phosphate

moiety of anatoxin-a(s), which will then lead to the release of hydroxy guanidine 9. One could

envision activation of the guanidine functionality could occur prior to serine hydroxyl addition to

help promote phosphorylation. It is this irreversible phosphorylation which leads to the build up

of acetylcholine in the synaptic cleft.17

This accumulation of acetylcholine leads to

neuromuscular paralysis and eventually death by respiratory failure. Symptoms of anatoxin-a(s)

poisoning include muscle weakness, respiratory distress, rhinorrhea, and eventually convulsions

followed by death.2

6

Scheme 1. Proposed mechanism of action of anatoxin-a(s).

In 1992, Moore proposed a biosynthesis of anatoxin-a(s) (Scheme 2). An arginine

molecule undergoes a stereoselective oxidation at the -position (10) followed by a nucleophilic

displacement by the guanidine functionality to form cyclic guanidine 11. Activation of the

hydroxy group to promote displacement probably occurs prior to nucleophilic displacement;

however, this exact mechanism is unknown at this time. Another methylene oxidation at the -

position (12) precedes a possible retro-aldol mechanism that releases a unit of glycine (13) and

aldehyde 14. Feeding experiments with two deuterium atoms incorporated at the C-3 position

and their subsequent replacement, gives some support to the proposed oxidation and retro-aldol

mechanism. However, by this proposed mechanism there should be a deuterium atom attached

to the aldehyde. The mechanism by which the dimethylamine portion is added to intermediate

14 to give 15 remains unknown. The proposed mechanism by Moore, involving a reductive

amination and dimethylation pathway, does not explain the loss of the remaining deuterium atom

that was observed during the feeding experiments. With the backbone framework in place,

tertiary amine 15 undergoes another selective oxidation (9) followed by phosphorylation,

probably by an enzymatic-mediated pathway, of the resulting hydroxyguanidine to provide

anatoxin-a(s) (8).18-21

To date, there is no information provided on the specific enzymes

involved in the biosynthesis of anatoxin-a(s).

7

Scheme 2. Biosynthesis of anatoxin-a(s) (8).

To determine the absolute stereochemistry of anatoxin-a(s), Moore prepared guanidine 15

(and its enantiomer) from L-asparagine (or D-asparagine, respectively) (Scheme 3). First,

carboxylic acid 16 was synthesized in two-steps from N-benzyloxycarbonyl-L-asparagine.22

Treatment of 16 with N-hydroxysuccinimide in the presence of DCC formed the N-

hydroxysuccinimide ester, which upon exposure to dimethyl amine gave amide 17. Next,

removal of the Boc group with TFA followed by Cbz deprotection with Pd/C and H2 gave the

corresponding diamine. Reduction of the amide with borane gave 18. Formation of the

guanidine 19 was accomplished by the treatment of 18 with S,S-dimethyl-N-

tosyliminodithiocarbonimidate in refluxing ethanol, which after removal of the toluenesulfonyl

8

group with HBr, gave guanidine 15. The circular dichroism (CD) spectrum of 15 matched the

CD spectrum of 15 that was generated from a natural source of anatoxin-a(s).

Scheme 3: Synthesis of guanidine 15.

1.1.2 Strategies for the formation of anatoxin-a(s)’s guanidine backbone: Inspiration

from related heterocycles.

The formation of nitrogen containing five-membered rings and related heterocycles can

be accomplished through metal-mediated methods, which provide an opportunity to control the

regioselectivity of the carbon-nitrogen bond forming event as well as the potential for reagent

controlled stereoselectivity.23

The challenge to synthesizing anatoxin-a(s) comes from the N-O

bond to be attached to the lone stereocenter present in the molecule. Two distinct strategies were

envisioned: formation of the carbon-nitrogen bond through a pre-tethered olefin or through a

diamination sequence (Scheme 4). For developing a synthetic strategy towards anatoxin-a(s), we

turned to the use of aminopalladation methods that have been developed for the construction of

guanidines, pyrrolidines, and ureas. A second strategy for the construction of 1,2-diamino

containing compounds is the diamination of olefins.

9

Scheme 4: Possible synthetic strategies for the synthesis of anatoxin-a(s).

One could envision two possible pathways for the amination of unactivated olefins

through palladium catalysis. The first pathway consists of cis-nucleopalladiation, or the

formation of a palladium-nitrogen complex (24), prior to the cyclization event (Scheme 5). The

second pathway involves the precoordination of palladium to the olefin (26), thereby activating it

towards nucleophilic addition and is generally referred to as trans-nucleopalladation. This

possibility of two different mechanistic pathways makes the development of an enantioselective

process difficult to achieve. Also, when applied to the construction of guanidines through a

similar transformation, the regioselectivity of the catalyst between the two available nitrogens

will play a key role in the outcome of the cyclization.

Scheme 5: Two possible pathways for nucleopalladation.

10

One strategy for the construction of nitrogen-containing heterocycles is through

palladium-catalyzed intramolecular ring closure of olefin containing aliphatic amines or related

nitrogen functionality. The Wolfe group in 2006 used this strategy for the synthesis of the natural

product (+)-preussin (Scheme 6).24

Treatment of amino-olefin 28 with phenylbromide in the

presence of the Pd(OAc)2/DPEphos catalyst system generated pyrrolidine 29 in 62% yield. A

likely mechanism involves oxidative insertion into the aryl bromide bond by Pd(0) followed by

formation of the aminopalladium complex. This complex then proceeds through a cis-

nucleopalladation pathway to give 29, after reductive coupling. This cyclization added the

flexibility to install various aryl groups at a late stage in order to probe the biological activity of

different (+)-preussin analogs. Pyrrolidine 29 was subjected to a one-pot reduction and

deprotection to afford (+)-preussin in 95% yield (96% ee).

Scheme 6. Synthesis of (+)-preussin.

The Wolfe group expanded on their pyrrolidine formation by optimizing the previous

reaction conditions for use in the construction of cyclic ureas.25

They treated arylbromide 30

with Pd2(dba)3/Xantphos, which undergoes oxidative insertion of the arylbromide bond (Scheme

7). The resulting palladium complex undergoes amidopalladation of allylic urea 31, followed by

nitrogen alkylation, and the formation of alkyl-palladium complex 32. This complex then

undergoes a reductive elimination to generate cyclic urea 33 in 88% yield and a 12:1 dr. The use

of base is necessary to promote amidopalladation. In 2012, they reported an enantioselective

11

variant of this methodology using the diastereomeric phosphoramidite ligand, (S)-Siphos-PE to

give 35 in 81% yield (92% ee).26

These examples by the Wolfe group all report a mechanism

that proceeds through cis-nucleopalladation mechanism and the formation of that complex is

facilitated by the presence of base.

Scheme 7. The synthesis of urea derivatives using the Pd2(dba)3/Xantphos catalyst system.

In 2011, the Stahl group reported their mechanistic studies of Wacker-type intramolecular

amination of alkenes (Scheme 8).27

Treatment of tosylamide 36 with Pd(OAc)2 in the presence

of pyridine produce pyrrolidine 38 in 87% yield. During the course of their mechanistic studies,

they observed that the rate of the reaction did not depend on the concentration of O2 but did show

a dependence on the concentrations of palladium catalyst and substrate. This observation

provided evidence toward a cis-aminopalladation pathway by which the mechanism would

proceed through likely intermediate 37. The Stahl group followed this work with a report in

2012 on an enantioselective variation of this reaction. Using the chiral pyridine oxazoline ligand

12

(S)-41 and Pd(TFA)2 they were able to form pyrrolidine 40 in 93% yield and 98% ee.

Scheme 8: Intramolecular amination of alkenes through aminopalladation.

Interestingly, they reported a low yield and almost a complete loss of enantioselectivity when

using Pd(OAc)2. They attributed this observation to the acetate ligands promoting a change in

the mechanistic pathway from a cis-nucleopalladation sequence to a trans-nucleopalladation

pathway. This is one of the few examples indicating the influence of the anionic ligands on the

mechanistic pathway of this type of cyclization.

A variation of the nucleopalladation of olefins is through the activation of the olefin by

the presence of a leaving group (typically carbonates) in the -position of the olefin. The Büchi

group reported in 1989 the use of this strategy in the synthesis of (±)-alchorneine and (±)-

isoalchorneine (Scheme 9).28

First, methoxy guanidine 42 was subjected to a Pd(0)-mediated

annulation that could proceed through either a -allyl Pd(II) intermediate or a cis-

amidopalladation complex to generate methoxy cyclic guanidine 43 in 81% yield. Methoxy

13

cyclic guanidine 43 was treated with two equivalents of PdCl2(CH3CN)2 in CH2Cl2 at 40 C to

give (±)-alchorneine in 46% yield.

Scheme 9. Synthesis of (±)-alchorneine.

In 1998, an enatioselective version of this strategy was applied by Trost as a method for

the desymmetrization of meso-alcohols. Treatment of a solution of allylic diol 44 and TsNCO in

THF with Pd2(dba)3/L-45 led to oxazolidin-2-one 48 in 84% yield and 99% ee (Scheme 10).29

A

significant enhancement of enantiomeric enrichment was observed upon the addition of an

external base, which has been shown to facilitate formation of the amidopalladium complex.

Scheme 10. Stereoselective formation of cyclic carbamates.

14

The reaction likely proceeds through dicarbamate 46, followed by formation of a cis-

amidopalladation complex (47). The regioselectivity of the cis-amidopalladation complex is

controlled through the use of chiral ligand L-45. The carbamate then cyclizes and, after

elimination of the carbamate, results in the stereoselective formation of oxazolidinone 48.

Another possibility would be the formation of a -allyl intermediate as the first-step, followed by

the formation a cis-amidopalladation complex. This complex would then undergo cyclization to

give 48.

In 1999, the Trost group applied this strategy to the asymmetric synthesis of the

cyanotoxin (–)-anatoxin-a by utilizing a similar -allyl Pd(II) complex to promote cyclization

(Scheme 11).30

Their end game strategy for the synthesis of (–)-anatoxin-a involved treatment of

allylic carbonate 49 with (dba)3Pd2CHCl3/L-51 to give [2.4.1] bicyclic amine 50 in 90% yield

and 88% ee. Pyrrolidine derivative 50 was then transformed into (-)-anatoxin-a.

Scheme 11. Asymmetric synthesis of (-)-anatoxin-a.

The Hirai group reported in 1997 the synthesis of (+)-prosopinine and (+)-palustrine

utilizing a key Pd(II)-mediated cyclization of urethanes.31

Urethane 52 was treated with

PdCl2(CH3CN)2 in THF to give fused piperdine 54 in 77% yield as a single diastereomer

(Scheme 12). The authors propose Pd(II) coordinates to the olefin (53), thereby activating it

towards nucleophilic addition. The oxygen of the methoxymethylether assists in the

15

organization of the transition state through the chelation of Pd(II). This is an example of a trans-

aminopalladation pathway, although it should be noted the authors do not provide any

experimental evidence for this pathway. Although the Hirai group used a similar strategy to

promote cyclization as the Trost group, the ability of these cyclizations to proceed through

different mechanistic pathways increases the difficulty of developing enantioselective conditions

for this cyclization. This difficulty will be compounded in the synthesis of guanidines due to the

presence of a second nitrogen that can participate in coordination with the metal catalyst.

Scheme 12. Synthesis of (+)-prosopinine.

In 2002, the Overman group developed a set of chiral Pd(II) catalysts to induce

asymmetric annulation of allylic ureas in the synthesis of oxazolines.32

They treated allylic urea

55 with 5 mol% of FOP-OTFA in 1:1 CH2Cl2/CH3NO2 to give urea 56 in 96% yield and 90% ee

(Scheme 13). FOP-OTFA is produced in situ by treating the corresponding Pd-I complex with

AgOTFA. Although a variety of silver salts were investigated, the best result was achieved with

silver trifluoroacetate. In 2005, a 2nd

generation catalyst was developed that replaced the

16

ferrocene ligand with a cobalt oxazoline palladacycle (COP) (Scheme 14).33

This catalyst

offered numerous advantages over the previous ferrocene based catalyst. The COP-catalyst was

not air sensitive and did not require pre-activation using silver salts. They treated allylic alcohol

57 with TsNCO in THF to generate the N-tosyl urea, followed by cyclization using COP-OAc in

CH2Cl2/AcOH (4:1) to give spirocycle 58 in 82% yield and 97% ee.

Scheme 13. Asymmetric intramolecular aminopalladation.

Scheme 14. Overman’s 2nd

generation catalysts.

A different approach to install 1,2-diamino functionality is through the diamination of

olefins. In 2005, the Muñiz group developed a diamination of terminal olefins that was

catalyzed by Pd(II).34

Urea 59 was treated with Pd(OAc)2 and PIDA in CH2Cl2 to give bicyclic

urea 60 in 89% yield (Scheme 15). To probe the reaction mechanism deuterated olefin 61 was

activated by Pd(II), followed by nucleophilic addition, afforded alkyl Pd(II)-species 62. Due to

the observed trans H-H relationship in the product, they proposed that PIDA oxidizes Pd(II) to

17

Pd(IV), and subsequent nucleophilic addition of the remaining secondary nitrogen with

concomitant loss of Pd(II) gives diamination product 63 as a single diastereomer.

Scheme 15. Diamination of terminal olefins and proposed pathway.

Muñiz followed their work on the cyclization of ureas by applying a similar strategy to

the synthesis of bicyclic guanidines in 2008.35

In their communication, they treated guanidine 64

with Pd(OAc)2 in the presence of Copper bromide to give bicyclic guanidine 65 in 99% yield

(Scheme 16). They did note that under these reaction conditions it was possible to isolate

substantial quantities of the related urea compound. Switching to copper chloride eventually

solved that issue. Also, the authors specifically only examined examples in which the two

nitrogens that had potential to cyclization first had the same substitution to avoid issues with

regioselectivity. There are few examples in the literature that address this issue of

regioselectivity in guanidine synthesis.

18

Scheme 16: Synthesis of bicyclic guanidine.

In 2007, the Shi group published a Pd(0)-catalyzed diamination of diene and trienes.36

The various dienes combined with di-tert-butyldiaziridinone (66) were treated with 10 mol% of

Pd(PPh3)4 in benzene at reflux to provide cyclic ureas in moderate to excellent yields (Table 1).

Both electron-rich and conjugated dienes were well tolerated and electron-deficient dienes gave

Table 1. Diamination of butadiene derivatives.

19

the desired cyclic ureas, albeit in a modest yield compared to electron rich dienes. When

conjugated trienes were used, the reaction took place at the central olefin selectively. One

noteworthy example involves the use of 2-trimethysilyloxybutadiene (67) to give the only

example of a monosubstituted cyclic urea in excellent yield (68, Table 1). The authors

proposethat the reaction first involves Pd(0) undergoing an oxidative insertion into the N-N bond

of di-tert-butyldiaziridinone followed by coordination of the diene to Pd(II) (Scheme 17). Next,

stepwise carbon-nitrogen bond formation generates the -allyl Pd(II) intermediate followed by

reductive elimination to regenerate Pd(0) and provide the product. Once formed these urea

compounds can be cleaved to reveal the diamino compounds that can be reclosed to form

guanidines.

Scheme 17. Shi's proposed mechanism for the diamination of olefins.

20

We wanted to explore two distinct pathways for the construction of anatoxin-a(s)’s

guanidine core. One pathway is through a nucleopalladation pathway that can cyclize onto

activated or unactived olefins. The challenges associated with the development of an

enantioselctive method for this cyclization include controlling a cis- or trans-nucleopalladation

pathway. Also, for this strategy to be successful for the synthesis of anatoxin-a(s), a

regioselective cyclization must be developed that can differentiate two distinct nitrogens. These

same challenges are also present in proceeding through a diamination strategy.

1.2 RESULTS AND DISCUSSION

Anatoxin-a(s) (8) is a structurally interesting organophosphate that contains a pentacyclic

guanidine moiety with a N-O bond attached to the only stereocenter. This functionality provides

a unique environment to expand on current metal-catalyzed carbon-nitrogen bond forming

methodology. Current methods focus on the formation of pyrrolidines and cyclic ureas through

various metal-catalyzed processes. However, there is a general lack of methodology for the

formation of cyclic guanidine rings with the most notable being the seminal work by Büchi28

and

the work of the Muñiz group on the Pd(II)-catalyzed formation of bicyclic guanidines,37

on

metal-catalyzed amidocarbonylations and amidoalkylations involving acyclic guanidine

precursors. The methodology developed by the Muñiz group relies on identical guanidine

nitrogens and does not address the regiochemistry in the cyclization. The ability to differentiate

between two electronically different nitrogens still remains a synthetic challenge. Also the

challenge of proceeding through a cis- or trans-nucleopalladation is a concern toward

establishing an enantioselective ring cyclization. Shi’s recently developed methodology for the

21

diamination of butadiene derivatives to give pentacyclic ureas would provide a facile route to

assemble the anatoxin-a(s) backbone.36

1.2.1 Route 1: Palladium(0)-catalyzed diaminations

The initial synthetic efforts towards the total synthesis of the cyanotoxin anatoxin-a(s) (8)

began by utilizing the diamination work developed by Shi (Table 1, vide supra). A

retrosynthetic analysis of anatoxin-a(s) resulted in silyl enol ether 68. Silyl enol ether 68 is

synthesized from di-tert-butyl-diaziridinone 66 and 2-trimethylsilyloxybutadiene 67 using Pd(0)

catalysis (Scheme 18).

Scheme 18. 1st Generation retrosynthetic analysis of anatoxin-a(s) (8).

To apply Shi’s diamination protocol to a synthesis of anatoxin-a(s), methyl vinyl ketone

was treated with TMSCl and triethylamine to give 2-trimethylsilyloxybutadiene 67 in 48% yield

(Scheme 19). Di-tert-butyl-diaziridinone 66 was synthesized from di-tert-butylurea according to

the procedure reported by Shi.38

2-Trimethylsilyloxybutadiene 67 and di-tert-butyldiaziridinone

66 were subjected to catalytic Pd(0) in benzene at 65 °C to give silyl enol ether 68. However,

22

Scheme 19. Attempted synthesis of silyl enol ether 68.

attempts at isolation of silyl enol ether 68 only resulted in desilylated ketone 70. To counteract

the instability of silyl enol ether 68, it was carried forward without purification. When subjected

to standard ozonolysis conditions, only decomposition of silyl enol ether 68 was observed.

Surmising that the difficulty with the subsequent ozonolysis was due to the inherent

instability of the TMS silyl enol ether, the more stable triethyl silyl enol ether was selected.

Methyl vinyl ketone in THF was subjected to a solution of LDA/ HMPA at -78 °C followed by

silylation of the enolate with TESCl and warming to 0 °C to provide 2-triethylsilyloxybutadiene

71 in 85% yield (Scheme 20). Next, a solution of 2-triethylsilyloxybutadiene 71 and di-tert-

butyldiaziridinone 66 in toluene or THF at 65 °C was treated with catalytic Pd(PPh3)4 to provide

TES enol ether 72 in 40-67% yield. With TES enol ether 72 in hand, conditions towards the

installation of the required nitrogen functionality were explored (Table 2). Ozone was bubbled

through a solution of TES enol ether 72 in CH2Cl2/CH3OH at -78 C but after treatment with

23

Scheme 20. Synthesis of TES silyl enol ether 72.

Me2S only decomposition of the starting material was observed. Treatment of 72 with aqueous

OsO4 in the presence or absence of NaIO4 in THF did not result in any dihydroxylated product or

carboxylic acid 69. Treatment of 72 with buffered m-CPBA in CH2Cl2 followed by the addition

of 1 M HCl in CH3OH gave -hydroxy ketone 73 in 5% yield (Entry 4, Table 2).39

In light of

the difficulties pertaining to the oxidation of silyl enol ether 72 and the modest yield of the

diamination reaction, an alternative route was chosen in which the backbone of anatoxin-a(s)

would be assembled through a key Pd(II) catalyzed amidocarbonylation reaction.

Table 2. Conditions for the oxidation of silyl enol ether 72.

24

1.2.2 Route 2: Palladium(II)-catalyzed amidocarbonylations

Previous work in the Wipf group involving amidocarbonylation reactions catalyzed by

Pd(II) was pursued by Gil Ma (Scheme 21).40

Working towards the development of new cyclic

urea derivatives as alternatives to DMPU, 4-bromo-1-butene was treated with neat methylamine

to give N-methyl-3-butenylamine. The resulting homoallyic amine was treated with phosgene to

give the corresponding chloroamidate, which was trapped with methylamine in the presence of

diisopropylethylamine to provide urea 74 in 66% yield. Urea 74 was subjected to PdCl2 (10 mol

%) and CuCl2 (3 equiv) in CH3OH under 1 atm of CO to generate alkyl-Pd(II) species 75,

followed by CO insertion, and trapping of the acyl Pd(II) intermediate with CH3OH to afford

methyl ester 76 in 89% yield. By revising our previous retrosynthetic scheme to utilize a similar

amidocarbonylation, one can envision that anatoxin-a(s) can be synthesized from methyl ester 77

(Scheme 22). Methyl ester 77 is the result of a Pd(II)-catalyzed amidocarbonylation of allylic

guanidine 78.

Scheme 21. Ma's work on DMPU derivatives.40

25

Scheme 22. 2nd

Generation retrosynthetic analysis of anatoxin-a(s) (8).

The synthesis of allylic guanidine 78 commenced with the treatment of allylamine with

CbzNCS41

to give thiourea 79 in 99% yield (Scheme 23). The electron-withdrawing nature of

the Cbz protecting group facilitated the formation of the guanidine functionality by increasing

the electrophilic nature of the thiourea. Treatment of thiourea 79 and H2NOBn•HCl with EDCI

gave allylic guanidine 78 in 76% yield. The use of H2NOBn•HCl serves three purposes: 1) The

strong electron donating nature of the amine facilitates guanidine formation, 2) to differentiate

the electronics of the two nitrogens for the key amidocarbonylation step, and 3) to install the

required N-O bond that is present in the natural product.

Scheme 23. Synthesis of allylic guanidine 78.

With access to guanidine 78, conditions for the key amidocarbonylation transformation

catalyzed by Pd(II) were investigated. Two requirements for this reaction were considered: the

regiochemistry had to be controlled and ligands for the development of an asymmetric variant

26

needed to be screened. Allyl guanidine 78 was treated with 10 mol% of Pd(OAc)2 and CuCl2 (3

equiv) in CH3OH under 1 atm of CO to give a 2.2:1 mixture of methyl esters 77 and 80 in a

combined 76% yield (Entry 1, Table 3). Other Pd(II) complexes gave complex mixtures of

Table 3. Amidocarbonylation conditions.

products that were not isolated. The addition of chiral ligands or other additives to affect the

isomeric ratio only succeeded in increasing the ratio of undesired regioisomer 77 without any

significant improvement in ee. Compounds 77 and 80 were assigned based on the chemical shift

of the C2-H of the Boc-derivatives 77-Boc and 80-Boc. By comparing the chemical shift of the

C2-H of 77-Boc ( 3.92-3.82) and C2-H of 80-Boc ( 4.49) and of comparable urea compounds

8142

and 82.43

Although the guanidine and urea compounds share groups with similar electronics

27

attached to the nitrogen adjacent to the stereocenter, the electronics between the guanidine

compounds and the ureas are different. This comparison was used as a preliminary structure

assignment while the synthesis was carried forward. Ideally a crystal structure would have been

obtained to unambiguously assign the structures but no crystal of these urea compounds or

derivatives were obtained.

Figure 6: Structure identification of cyclization compounds and comparable urea compounds 81 and 82.

The amidocarbonylation is thought to proceed through a Wacker-type cyclization

pathway outlined below (Scheme 24).44

First, Pd(II) may coordinate to olefin 83, thereby

activating it towards nucleophilic addition, or, alternatively, Pd(II) forms N-Pd(II)amido species

84 with concomitant loss of HX. Next, amidoalkylation forms alkyl-Pd(II) species 85, followed

by CO insertion to give acyl-Pd(II) species 86. Subsequent reductive elimination forms methyl

ester 77 and Pd(0), which is reoxidized by CuCl2 to Pd(II). Due to the prevalence of the

undesired methyl ester, by addition of the weaker nucleophile, it is thought that the reaction

proceeds through amido-Pd(II) species 83 and not by simple Lewis activation of the olefin 84.

28

Scheme 24. Proposed mechanism of amidocarbonylation.

Despite the preliminary results of the amidocarbonylation giving the undesired

regioisomer, it was decided to proceed with the hydrolysis of the methyl esters; however, when a

solution of methyl esters 77 and 80 were subjected to KOTMS in THF, only carboxylic acid 87

was observed in 55 – 69% yield (Scheme 25). Initially, KOTMS deprotonates to the methyl

ester of regioisomer 80, followed by ring opening to form the ,-unsaturated ester 89. The ring

opening of ester 80 is facilitated by the electron withdrawing nature of the Cbz-protecting group.

At this juncture, the negative charge is delocalized between N1 (89) and N2 (90). N2 then adds

back into the --unsaturated ester to give the desired methyl ester 77. Calculations show that

77 is the thermodynamically more stable compound by 5.3 Kcal/mol.* The isomerization is

followed by hydrolysis.

* Calculations preformed using MMFF conformer distribution followed by PM3 equilibration of geometry using Spartan 10

29

Scheme 25. Synthesis of carboxylic acid 87 and proposed mechanism of ester isomerization.

With a route to carboxylic acid 87, conditions to install the required nitrogen

functionality by a Curtius rearrangement were explored. 45

Exposing of carboxylic acid 87 to

DPPA and triethylamine in tert-BuOH at reflux resulted in decomposition of starting material

(Entry 1, Table 4). In a more stepwise approach towards formation of the acyl azide, carboxylic

acid 87 was treated with oxalyl chloride followed by the addition of sodium azide. To the

resulting solution was added tert-BuOH and the reaction mixture was heated at reflux; however,

only decomposition of starting material was observed (Entry 2, Table 4). In 2005, the Lebel

group reported a mild one-pot procedure for the synthesis of Boc-protected amines by treating

carboxylic acids with sodium azide, Boc2O, and TBAB in the presence of Zn(OTf)2 via a Curtius

rearrangement.46

Unfortunately, when carboxylic acid 87 was exposed to these conditions, no

reaction of the starting material was observed (Entry 3, Table 4). Due to the unforeseen

difficulty in accomplishing the Curtius rearrangement, an alternative method for the installation

of the nitrogen functionality on the side chain was examined.

30

Table 4. Conditions for attempted Curtius rearrangement of carboxylic acid 87.

In 2008, the Renaud group reported the synthesis of alkyl azides using the radical

decarboxylation of thiohydroxamate esters.47

These conditions were applied to the coupling of

N-hydroxydithiocarbamates 92 and 93 with carboxylic acid 87 in the presence of DCC and

DMAP. The corresponding thiohydroxamate esters 94 and 95 were produced in 76% and 56%

yield, respectively (Scheme 26). Unfortunately, exposing thiohydroxamate esters 94 and 95 to

AIBN in benzene at reflux in the presence of phenyl sulfonylazide slowly decomposed the

thiohydroxamate esters and failed to give desired alkyl azide 96. A possible explanation is that

upon the generation of the primary radical, a 1,2-H migration occurs to produce a more stable

tertiary radical or a ring-opening event occurs to produce an N-centered radical that subsequently

decomposes.

31

Scheme 26. Radical decarboxylation of thiohydroxamate esters 94 and 95.

After the radical decarboxylation failed to provide alkyl azide 96, a Hofmann

rearrangement was explored for the installment of the required nitrogen functionality.48,49

A

mixture of methyl esters 77 and 80 in CH3OH at -78 C was treated with liquid NH3 to give

amide 97 in 64% yield (Scheme 27). In the presence of 5 equiv of Pb(OAc)4 and triethylamine

in tert-BuOH/ DMF at 100 C none of desired rearranged Boc-protected amine 97 was observed

(Table 5). Switching Pb(OAc)4 to PIFA only resulted in decomposition of starting material.

Scheme 27. Formation of amide 97.

32

Examination of the less active PIDA led to similar results (Entry 3, Table 5). Due to the

troubling preliminary results of the key amidocarbonylation cyclization and the difficulty of

installing the required nitrogen functionality, an alternative route that utilized a Pd(II)-mediated

amidoalkylation was pursued.

Table 5. Conditions for attempted Hofmann rearrangement of amide 97.

1.2.3 Route 3: Palladium(II)-catalyzed amidoalkylations

Our previous retrosynthetic analysis was revised toward an approach to anatoxin-a(s) from olefin

98. Olefin 98 can be prepared by a Pd(II)-catalyzed amidoalkylation of allylic guanidine 99

(Scheme 28). Mono protection of commercially available 2-butenediol using TBDPSCl in the

33

Scheme 28. 3rd

Generation retrosynthetic analysis of anatoxin-a(s) (8).

presence of imidazole gave (Z)-4-(tert-butyldiphenylsilyloxy)but-2-en-1-ol in 94% yield

(Scheme 29).50

Next, bromide displacement of the free alcohol with Br2, PPh3 and imidazole in

THF afforded the allylic bromide in 91% yield.51

The allylic bromide was displaced with

PhthNK in DMF at 80 C to provide phthalamide 100 in 75% yield. Phthalimide 100 was

carried through a two-step procedure of phthalimide removal with hydrazine in EtOH at reflux

and trapping the resulting allylic amine with CbzNCS41

in CH2Cl2 to yield thiourea 101 in 96%

yield over the two steps. Thiourea 101 was coupled with H2NOBnHCl in the presence of

diisopropylethylamine and EDCI to afforded guanidine 102 in 71% yield (Scheme 30). The

guanidine was subjected to a two-step procedure to install the benzyl moiety required for the key

Scheme 29. Synthesis of thiourea 101.

34

amidoalkylation step. The guanidine was treated with TBAF in THF followed by the addition of

BzCl to the crude alcohol in the presence of pyridine and DMAP to provide guanidine 99 in 22%

yield over the two steps. It is unclear why the isolated yield over the two steps was low, but

perhaps exposing 102 to unbuffered TBAF led to cleavage of the Cbz group. Unfortunately no

side-products were isolated that would provide insight on this issue. Due to the poor yield for

Scheme 30. Synthesis of guanidine 99.

guanidine 99, the synthetic sequence was changed. First, thiourea 101 was deprotected with

TBAF, followed by benzoylation of the resulting allylic alcohol to give thiourea 103, and

guanidine formation with H2NOBnHCl in the presence of diisopropylethylamine and EDCI to

afford guanidine 99 in 38% yield over three steps.

With guanidine 99 in hand, suitable conditions to effect the key amidoalkylation

catalyzed by Pd(II) were explored. Initially, treatment of guanidine 99 with 20 mol% of

PdCl2(CH3CN)2 in THF (0.05 M) followed by Boc protection of the free amine gave olefin 104

35

in 59% yield over the two steps (Entry 2, Table 6). The subsequent Boc protection was due to

the unforeseen polarity of the unprotected compound. Although compound 98 behaves normally

Table 6. Screening conditions for key amidoalkylation of guanidine 99.

36

by TLC analysis, purification of 98 by chromatography on SiO2 required a substantially more

polar mobile phase than was required for TLC analysis. Increasing the concentration of the

initial Pd(II)-mediated cyclization reaction resulted in an increased 79% yield of olefin 104. The

use of a Pd(0) catalyst resulted in a poor 17% yield of olefin 105 (Entry 1, Table 6). Using the

palladacycle catalyst developed by Overman (Scheme 14),33

a 1:1 ratio of 104 and 105 in a 70%

combined yield with olefin 104 in 48% ee was obtained. The use of Josiphos ligand A

completely shut down the catalytic cycle (Entry 6, Table 6).52

Subjecting guanidine 99 to

PdCl2(CH3CN)2/pyBox B catalyst system in 1,2-dichloroethane at 85 C gave the desired olefin

in 67% yield and 2% ee (Entry 8, Table 6). Calculations show that isomer 104 is

thermodynamically more stable than 105 by 1.91 Kcal/mol.†

In 2002, the Overman group proposed a mechanism for a similar transformation

involving the cyclization of carbamate derivatives.32

The Pd(II) initially coordinates to olefin 99,

directed by chelation from the benzoyl carbonyl oxygen, and activating the olefin towards

intramolecular nucleophilic attack (106). An intramolecular nucleophilic addition of the more

electron rich nitrogen of the guanidine moiety and release of HCl forms alkyl-Pd(II) intermediate

107. Finally, elimination of olefin 98 regenerates Pd(II) that can re-enter the catalytic cycle

(Scheme 31).

Facilitated by a route to racemic olefin 104, the completion of the synthesis of anatoxin-

a(s) was pursued. A solution of olefin 104 in CH3OH/CH2Cl2 buffered with 10 equiv of pyridine

was cooled to -78 C (Scheme 32). Ozone was bubbled through the solution followed by

treatment with NaBH4 to afford alcohol 108 in 61% yield. In the absence of pyridine only

† Calculations preformed using MMFF conformer distribution followed by PM3 equilibration of geometry using Spartan 10

37

Scheme 31. Proposed catalytic cycle of amidoalkylation.

decomposition of starting material was observed. A recent report from Dussault hypothesizes

that pyridine could potentially add to the carbonyl oxide that is formed after the break down of

the initial ozonide to generate intermediate 109.53

The zwitterionic adduct 109 then adds to a

second equivalent of carbonyl oxide (110). Then, decomposition of 111 would produce

2equivalents of the aldehyde (112) and release an equivalent of O2 and pyridine. The authors

observed a general trend of increased yields when pyridine was added to these reactions.

Alcohol 108 was mesylated with MsCl and triethylamine in CH2Cl2 followed by treatment of the

crude mesylate with sodium azide in DMF at 65 C to give alkyl azide 113 in 77% yield over the

two steps.

38

Scheme 32. Synthesis of alkyl azide 113.

With a synthesis of azide 113 realized, conditions for reduction and subsequent

dimethylation were investigated (Scheme 33). Initially, several conditions were explored to

reduce the azide to the amine. Either Pd/C and H2 or standard Staudinger conditions (not shown)

gave the corresponding amine quantitatively. Surprisingly, the subsequent dimethylation of the

free amine proved difficult. Several conditions were surveyed to achieve the dimethylation.

Mostly variations of the Eschweiler-Clarke conditions utilizing formaldehyde and NaCNBH3

were tried. However, only the primary amine was reisolated. A possible explanation for this

difficulty is the ability of the guanidine nitrogens or the O-benzyl oxygen to form hydrogen

bonds to the free amine and preventing nucleophilic addition into formaldehyde. A Merck

Molecular Force Field (MMFF) lowest energy conformation calculation of the primary amine

showed the presence of hydrogen bonding with the N-OBn oxygen. Accordingly, azide 113 was

reduced by Pd/C and H2 with in situ trapping of the resulting primary amine with di-tert-

butyldicarbonate to give tert-butylcarbamate 115 in 62% yield. Methylation of tert-

39

butylcarbamate 115 was achieved in 79% yield by treating a solution of tert-butylcarbamate 115

and 20 equiv of iodomethane in DMF at -20 C with NaH and warming to room temperature to

give methylcarbamate 116.

Scheme 33. Synthesis of methyl carbamate 116.

Methyl carbamate 116 was subjected to a solution of CH2Cl2/TFA (4:1) to remove both

Boc groups followed by a selective methylation of the secondary amine in the presence of the

exposed guanidine nitrogen under Eschweiler-Clarke conditions to afford dimethylamine

117.54,55

Treatment of 117 with Pd/C and H2 at 60 psi gave an inseparable mixture of N-hydroxyl

guanidine 9 and guanidine 118 in 35% and 56% yield, respectively, over the three steps. The 1H-

NMR spectra of 9 and 118 matched the values for those compounds that were reported in the

isolation paper (Scheme 34).17

The original structures were assigned based on enriched 50% 13

C

and 90% 15

N, which was used to confirm the guanidine core of anatoxin-a(s). The structural

assignment was supported by FAB MS-MS and high-resolution data. The location of the

hydroxyl group was determined based on similar chemical shifts observed in neosaxitoxin and

40

saxitoxin. Also, Moore and coworkers prepared synthetic 15 (the single enantiomer of 118,

Scheme 3), which matched the spectral data of 15 that had been obtained from degradation of

anatoxin-s(s).

Scheme 34. Synthesis of N-hydroxy guanidine 9.

Since the major product under the only conditions that succeeded in removing the benzyl

group was 118, we decided to exchange the benzyl group with a more labile functionality. To

that extent, starting with thiourea 101, the synthetic route would be optimized and the benzyl

group would be replaced with a functionality that would be more amendable to a late-stage

cleavage.

Due to the low yields in the formation of the cyclization precursors using TBAF for silyl

deprotection, we switched to the milder HF•pyr. Benzoylation of the alcohol produced thiourea

103 in 82% yield over the two steps (Scheme 35). Following our previously optimized

conditions for guanidine formation, treatment of thiourea 103 with benzyl hydroxyl amine in the

presence of diisopropylethylamine gave benzyl derivative 99 in 74% yield. Initially several silyl

groups (TES, TBS, TBDPS) and THP were tried in place of the benzyl moiety but after

41

cyclization, the substrate was too labile to be viable. To install the methoxy methyl

functionality, we first treated commercially available hydroxy phthalimide 119 with freshly

prepared chloromethyl methyl ether to give methoxy methyl protected hydroxy phthalimide 120.

Treatment of 120 with hydrazine gave MOM-protected N-hydroxy amine, which when added to

a solution of 103 in the presence of EDCI and diisopropylethylamine, gave guanidine 121 in

81% yield.

Scheme 35. Improved synthesis of 99 and synthesis of MOM derivative 121.

Next, subjecting guanidine derivative 121 to our previously optimized cyclization

conditions with PdCl2(CH3CN)2 gave a single spot by TLC. After Boc-protection of the exposed

guanidine nitrogen, olefin 122 was isolated in 95% yield as the sole regioisomer (assigned based

on the chemical shift of the C2-H) (Table 7). During the cyclization studies with benzylated

guanidine derivative 99, treatment of 99 with Overman’s chiral palladium(II) catalyst, COP-

OAc. Unfortunately, when 99 was subjected to those conditions, a mixture of regioisomers was

42

observed. After SFC separation of the regioisomers, a 3:1 e.r. was observed by chiral SFC. For

MOM derivative 121 we switched to the (S)-(+)-COP-Cl catalyst and observed only a single

regioisomer of the cyclization products (122 or 123) was observed. Analysis by chiral SFC

showed that isomer 122 was produced in a 3:1 e.r. This effect of the anionic ligand

Table 7. Pd(II)-mediated cyclization of MOM-derivative 121.

influencing the outcome of the reaction is similar to what was reported by Stahl for the

cyclization of aliphatic amines to generate pyrrolidine compounds (Scheme 36).56

This effect

was attributated to a change in the mechanistic pathway from a trans-aminopalladation pathway

to a cis-aminopalladation pathway (Scheme 5). In their reported cyclization with Pd(TFA)2, a

90% yield (96% ee) and a 9:1 ratio of trans- vs. cis-aminopalladation (AP) products was

observed; however, when Pd(OAc)2 was used as a catalayst a decrease in yield,

enantioselectivity (48%, 20% ee), and a reversal of the ratio of trans- vs. cis-AP products (1:9)

43

was observed. Interestingly, separating the trans products (125 and 126) still showed that these

compounds were produced in a high enanteomeric ratio. They concluded that the poor

enantioselectivity they observed was for the cis-aminopalladation pathway, while high

enantioselectivities were observed for the trans-aminopalladation pathway.

Scheme 36: Trans-aminopalladation (AP) vs. cis-aminopalladation.

NHTs

D

Ph

PdIIX2 / LNTs

NMe

N

O

Ph

L

Pd(TFA)2: 93%, 98% ee (9:1 trans/cis)

Pd(OAc)2: 48%, 20% ee (1:9 trans*/cis)

trans* (9:1 e.r.)

3Å M. S., toluene

H Ph

+

NTs

D Ph

NTs

D Ph

NTs

H Ph

+ +

trans-AP products cis-AP products

125 126 127 128124

1 atm O2

With a route established for the production of 122 and conditions for a moderately

enantioselective cyclization developed, completion of a synthesis of anatoxin-a(s) was pursued.

To achieve that we bubbled ozone through a solution of 122 that had been buffered with 10

equiv of pyridine, as we had determined to be required in the previous route (Scheme 37). After

treatment of the ozonide intermediate with NaBH4 alcohol 129 was isolated in 78% yield. Next,

mesylation of the alcohol with methane sulfonyl chloride, followed by nucleophilic displacement

of the mesylate with NaN3 gave alkyl azide 130 in 87% yield over the two steps. When azide

130 was reduced under the previous conditions for azide reduction (Pd/C, H2) lower and

irreproducible yields were observed. However, by switching to Lindlar’s catalyst wesaw an

improved and reproducible 80% yield of carbamate 131. Presumably, the lower yield was due to

44

competitive cleavage of the Cbz group, which was not observed when utilizing the less reactive

Lindlar’s catalyst. With an efficient route to carbamate 131 established, a two-step procedure to

install the dimethyl amine functionality was applied. Methylation of the carbamate with CH3I

and NaH gave, after exposure to TFA, methyl amine 132 in 73% yield over the two steps.

Scheme 37. Synthesis of hydroxy guanidine 134.

Exposing methyl amine 132 to reductive amination conditions produced dimethyl amine

133 in 66% yield. Cleavage of the methoxy methyl functionality proceeded smoothly to give

hydroxy guanidine 134 in 74% yield. With the successful cleavage of the methoxy methyl group

we next explored conditions for the successful phosphorylation of 134.

Before exploring phosphorylation conditions, a model system of 134 was developed as

phosphorylation of 134 would not be trivial. Towards that end commercially available ethylene

thiourea (135) was treated with HI in CH3OH to give alkylated thioimidate 136 in 93% yield

(Scheme 38). Installation of the Cbz group proceeded smoothly by treating 136 with CbzCl and

45

triethylamine to give 137 in 99% yield. As was worked out during the synthesis of 134, the Cbz

group is installed first to facilitate the addition of the electron rich nitrogen functionality. To

finish the synthesis of the guanidine core, 119 was exposed to hydrazine and the resulting amine

was added to a solution of 137, which after treatment with AgNO3 gave guanidine 138 in 74%

yield. Finally, removal of the MOM group was achieved using TMSBr to give our desired

model system 139 in 99% yield.

Scheme 38. A model system synthesis of 134 to explore phosphorylation conditions.

With a facile synthesis of the model system achieved, hydroxyl guanidine 139 was

subjected to conditions to install the biologically important phosphate moiety. The most

common conditions for the installation of phosphate groups typically involve the use of

chlorophosphates as the phosphorylating agent. Initially, 139 was treated with dimethyl

chlorophosphate in CH3CN with triethylamine and 140 was isolated in a 31% yield (Entry 1,

Table 8). Efforts to improve this yield were initially focused on utilizing different bases. First,

deprotonation of 139 with NaH at 0 oC, followed the addition of dimethyl chlorophosphate and

warming to rt did not yield 140. Switching to NaHMDS we were able to isolate 140 in a similar

33% yield (Entry 3, Table 8) but no product was detected when KHMDS was used. A decrease

46

in the isolated yield of 140 was observed when DBU was used as the base (Entry 5, Table 8) and

the use of diisopropylethylamine did not produce phosphorylated guanidine 142. Interestingly,

the use of diphenyl chlorophosphate was observed to give a slightly higher yield under the initial

conditions, which may be evidence that the low yield was due to the stability of dimethyl

chlorophosphate (Entry 7, Table 8).

Table 8: Exploration of different bases to optimize phosphorylation.

With no observable improvement to the phosphorylation when utilizing different bases,

solvent effects on the phosphorylation of 139 were explored. It was known at this time that

hydroxyguanidine compound 134 was not soluble in CH3CN, which added another potential

hurdle to the success of this reaction. Using our initial phosphorylation conditions, while

increasing the equiv of dimethyl chlorophosphate from two to five equivalents, we observed a

47

37% yield of 140 (Entry 1, Table 9). Unfortunately, using these same conditions with different

solvents (DMF, NMP, PhCN) resulted in no observable product formation.

Table 9: Solvent screen under the phosphorylation conditions.

With a small variation of yield observed dependent on the equivalents of the

phosphorylation agent used and no improvement of the reaction utilizing different solvents,

different phosphorylating reagents were explored. First, a coupling strategy was explored by

treating 139 with dimethyl phosphate in the presence of diisopropyl carbodiimide (DIC) (Entry

1, Table 10). Under these conditions no observable product was detected and the starting

material had disappeared by LC-MS analysis of the reaction mixture (Entry 1, Table 10). Next, a

more reactive phosphorylating agent than a chlorophosphate was used. To this end, a solution of

48

Table 10: Exploring different phosphorylation conditions.

dimethyl phosphate and triethylamine was treated with TFAA, followed by the addition of

methyl imidazole, which generated the highly active dimethyl methyl-imidazole phosphate. The

addition of this phosphorylating agent to 139 produced the desired dimethyl phosphate 140 in an

improved 49% yield (Entry 2, Table 10). Attempts at using phenyl dichlorophosphate followed

by the addition of methanol (Entry 3, Table 10) and the addition dibenzyl

diethylphosphoramidite to 139 followed m-CPBA did not yield did not yield any observable

product formation (Entry 4, Table 10).

Hydroxy guanidine 134 was subjected to the two phosphorylation conditions that had

been successful on the model system (139) (Scheme 39). Unfortunately, subjecting 134 to

dimethyl chlorophosphate in the presence of triethylamine did not lead to successful

phosphorylation. Exposure of 134 to the more active dimethyl methylimidazole phosphate did

not result in any detectable (by TLC, 1H-NMR, or LC-MS) amount of 135.

49

Scheme 39: Attempted phosphorylation of 134.

Unable to achieve phosphorylation of 134 under our previously worked out conditions,

we proceeded to try other methods of phosphorylation. Subjecting 134 to dimethyl

chlorophosphonate in a variety of solvents and different bases did not produce any observable

trace of 134. Exposure of 134 to either dibenzyl N,N-diethyl phosphoramidite or dimethyl N,N-

diethyl phosphoramidite in the presence of triethylamine resulted in the intermediate mass

potentially being detected by direct inject mass spectroscopic analysis of the reaction solution

([M + H]+). No

1H-NMR data of this intermediate could be obtained. The addition of m-CPBA

to the solution resulted in what appeared to be oxidation of the intermediate to the mass expected

for 140/141 as detected by LC-MS analysis of the reaction solution. Unfortunately, this mass

was never detected after work-up of the reaction mixture. Without spectroscopic proof of the

intermediate or the resulting product, it is uncertain what occured under these conditions. Also,

attempts at using a coupling strategy involving DCC and dimethyl phosphate resulted in loss of

the starting material with no detectable product formation. In 2004, the Jones group published a

procedure for the phosphorylation of alcohols utilizing N-phosphoryl oxazolidinones as transfer

reagents.57,58

Under their conditions, they are able to achieve phosphorylation of primary,

secondary, tertiary, and aromatic alcohols. Unfortunately, under their reported conditions we

saw no detectable phosphorylation of 134. The use of a harder base to promote anion formation

also did not yield 140.

50

1.3 CONCLUSION

Anatoxin-a(s) (8) is a unique organophosphate and one of the strongest PNS-anticholinesterase

agents. The backbone framework was assebled using an array of Pd(0) and Pd(II) metal-

mediated reactions. The Initial effort towards the total synthesis of anatoxin-a(s) centered around

assembling the heterocyclic scaffold using Shi’s diamination procedure of 2-trimethylsilyloxy-

butadiene (67) to give silyl enol ether 68 (Scheme 19, vide supra); however, difficulties in

functionalizing silyl enol ether 68 led to different methods to construct the framework of

anatoxin-a(s) being explored. A key amidocarbonylation reaction catalyzed by Pd(II) was

pursued but preliminary results showed that the undesired regioisomer was the preferred product.

Also, as in the 1st generation route, installation of the dimethylamine side-chain present in the

natural product proved difficult. A 3rd

generation route involved a key amidoalkylation of

guanidine 121, which led to advanced intermediate 134 in 18 steps and 8.0% overall yield. In

this route, a new methodology for the regioselective cyclization of unsymmetrical guanidines

was developed. Using the catalyst system developed by Overman, (R)-(–)-COP-Cl, we were

able to achieve a 3:1 enantiomeric ratio for the key palladium-mediated cyclization sequence.

Although a variety of conditions were attempted to phosphorylate 134, none of the conditions

tried were successful in generating phosphate 140.

51

2.0 EXPERIMENTAL

General. All glassware was dried in an oven at 140 C for 2 h or flame dried prior to use and all

moisture-sensitive reactions were performed under a dry N2 atmosphere unless otherwise stated.

THF and Et2O were distilled from sodium/benzophenone ketyl; triethylamine, pyridine, and

diisopropylethylamine were distilled from CaH2 and stored over KOH; DMF was through

activated neutral alumina and dried for 4 days over 3 Å molecular sieves; CH2Cl2 and toluene

were purified by passage through an activated alumina filtration system; 1,2-dichloroethane was

distilled over CaH2; CbzNCS,41

di-tert-butylurea,38

66,38

67,47

71,47

(Z)-4-(tert-

Butyldiphenylsilyloxy)but-2-en-1-ol,50

(Z)-(4-Bromobut-2-enyloxy)(tert-butyl)diphenylsilane,51

were prepared according to literature procedures.

Reactions were monitored by thin-layer chromatography using pre-coated silica gel 60

F254 plates (EMD, 250 µm thickness) and visualization was accomplished with a 254 nm UV

light and by staining with a KMnO4 solution (1.5 g of KMnO4 and 1.5 g of K2CO3 in 100 mL of

a 0.1% NaOH solution) or Vaughn’s reagent (4.8 g of (NH4)6Mo7O24•4 H2O and 0.2 g of

Ce(SO4)2 in 100 mL of a 3.5 N H2SO4 solution). Flash chromatography was performed using

SiO2 (Silicycle, Silia-P Flash Silica Gel, 40-63 µm) or Florisil (purchased from Fischer

chemical and used as received, 100 - 200 mesh).

52

1H and

13C NMR spectra were obtained on a Bruker advance 300 MHz in CDCl3 unless

otherwise noted. Chemical shifts (δ) were reported in parts per million with the residual solvent

peak used as an internal standard δ 1H /

13C (Solvent); 7.26 /77.23 (CDCl3) and are tabulated as

follows: chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, dd = doublet of

doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublet of doublets,

dt = doublet of triplets, t = triplet, td = triplet of doublets, bt = broad triplet, tt = triplet of triplets,

at = apparent triplet, q = quartet, m = multiplet), number of protons, and coupling constant(s).

Infrared spectra were determined as neat solids or oils on a Smiths Detection IdentifyIR FT-IR

spectrometer. Mass spectra were obtained on a Micromass Autospec double focusing

instrument. Melting points were determined using a Laboratory Devices Mel Temp II in open

capillary tubes and are uncorrected. Enantiomeric ratios were determined using a Mettler Toledo

AG – Berger SFCTM

Analytix.

1,3-Di-tert-butyl-4-(1-(trimethylsilyloxy)vinyl)imidazolidin-2-one (68)36

and 4-acetyl-1,3-di-

tert-butylimidazolidin-2-one (70). A solution of 66 (0.500 g, 2.94 mmol) in toluene (8.80 mL)

was treated with Pd(PPh3)4 (0.339 g, 0.294 mmol). To the mixture was added 67 (1.67 g, 5.87

mmol) and the resulting solution was heated to 65 ˚C for 1 h. The reaction solution was cooled

53

to rt and filtered through SiO2 (9:1 hexanes/EtOAc) to give 68 (174 mg, 19 %): 1H NMR (300

MHz, benzene-d6) δ 4.33 (d, 1 H, J = 1.5 Hz), 4.07 (d, 1 H, J = 1.5 Hz ) 3.71 (dd, 1 H, J = 3.0,

9.0 Hz), 3.10 (at, 1 H, J = 8.4, 9.0 Hz), 3.00 (dd, 1 H, J = 3.0, 8.4 Hz), 1.51 (s, 9 H), 1.34 (s, 9

H), 0.15 (s, 9 H); and 70: IR (ATR) 1718, 1679 cm-1

; 1H NMR (300 MHz, benzene-d6) δ 3.72

(dd, 1 H, J = 4.8, 10.2 Hz), 2.85 (dd, 1 H, J = 9.0, 9.9 Hz ) 2.54 (dd, 1 H, J = 4.5, 9.0 Hz), 1.83

(s, 3 H), 1.28 (s, 9 H), 1.20 (s, 9 H); 13

C NMR (75 MHz, benzene-d6) δ 208.3, 160.6, 61.6, 54.5,

53.4, 43.7, 28.5, 27.5, 24.5; HRMS (ES+) m/z calcd for C13H25N2O2 ([M + H]

+) 241.1916, found

241.1915.

1,3-Di-tert-butyl-4-(1-(triethylsilyloxy)vinyl)imidazolidin-2-one (72). A solution of 66 (50.0

mg, 0.294 mmol) and Pd(PPh3)4 (33.9 mg, 0.0294 mmol) in THF (0.100 mL) was treated with 71

(108 mg, 0.587 mmol) and heated to 65 ˚C for 1 h. The reaction solution was cooled to room rt

and filtered through Florisil (9:1 hexanes/EtOAc) to give 72 (48.1 mg, 46%): IR (ATR) 1690

cm-1

; 1H NMR (300 MHz, benzene-d6) δ 4.26 (d, 1 H, J = 1.8 Hz), 4.06 (d, 1 H, J = 1.5 Hz ),

3.74 (dd, 1 H, J = 3.3, 9.3 Hz), 3.11 (dd, 1 H, J = 8.4, 9.3 Hz), 3.02 (dd, 1 H, J = 3.0, 8.1 Hz),

1.51 (s, 9 H), 1.35 (s, 9 H), 0.98 (s, 9 H), 0.65 (m, 6 H); 13

C NMR (75 MHz, CDCl3) δ 160.9,

54

160.3, 88.3, 55.7, 54.0, 52.9, 47.2, 28.7, 27.6, 6.9, 5.0; HRMS (ES+) m/z calcd for

C19H38N2O3SiNa ([M + Na]+) 377.2600, found 377.2590.

1,3-Di-tert-butyl-4-(2-hydroxyacetyl)imidazolidin-2-one (73). A solution of 72 (109 mg,

0.307 mmol) in CH2Cl2 (1.5 mL) and 5% aq. NaHCO3 (1.5 mL) was cooled to 0 ˚C, treated with

m-CPBA (151 mg, 0.613 mmol), stirred at rt overnight. The reaction was diluted with sat.

NaHCO3 and extracted with CH2Cl2. The combined organic extracts were washed with sat.

Na2SO3, sat. NaHCO3, brine, dried (Na2SO4), and concentrated. The crude residue was

dissolved in CH3OH (1.0 mL) and treated with 1.5 M HCl (1.0 mL). The reaction mixture was

stirred overnight and extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were

washed with water, dried (Na2SO4), concentrated, and purified by chromatography on SiO2 (1:1

hexanes/EtOAc) to give 73 (3.9 mg, 5%): IR (ATR) 3396, 1702 cm-1

; 1H NMR (300 MHz,

benzene-d6) δ 4.35 (dd, 1 H, J = 5.1, 19.8 Hz), 4.13 (dd, 1 H, J = 4.8, 19.8 Hz ) 3.72 (dd, 1 H, J =

4.2, 9.9 Hz), 2.86 (t, 1 H, J = 5.1 Hz), 2.74 (t, 1 H, J = 9.6 Hz), 2.44 (dd, 1 H, J = 4.2 9.3 Hz),

1.18 (s, 9 H), 1.13 (s, 9 H); HRMS (EI+) m/z calcd for C13H24N2O3 ([M]

+.) 256.1787, found

256.1779.

55

1-Benzyloxycarbonyl-3-allylthiourea (79). To a solution of CbzNCS41

(0.831 g, 4.30 mmol) in

CH2Cl2 (164 mL) at 0 C was added allylamine (0.330 mL, 4.30 mmol). The solution was

warmed to rt, stirred for 1.5 h, and quenched with 1 M HCl (10.0 mL). The organic layer was

washed with water (10 mL) and brine (10 mL), dried (Na2SO4), concentrated, and purified by

chromatography on SiO2 (4:1, hexanes/EtOAc) to give 79 (1.07 g, 99%) as a white solid: Mp

61.3-63.5 C; IR (ATR) 3252, 3164, 1715, 1534 cm

-1.

1H NMR (300 MHz, CDCl3) 9.71 (s, 1

H), 8.11 (s, 1 H), 7.43–7.33 (m, 5 H), 5.92 (dddd, 1 H, J = 5.7, 5.7, 10.5, 17.1 Hz), 5.32–5.21 (m,

2 H), 5.18 (s, 2 H), 4.30 (tt, 2 H, J = 1.2, 5.7 Hz); 13

C NMR (75 MHz, CDCl3) 179.3, 152.7,

134.7, 132.1, 128.9, 128.8, 128.3, 117.7, 68.2, 48.0; HRMS (EI+) m/z calcd for C12H14N2O2S

([M]+.

) 250.0776, found 250.0786.

56

1- Benzyloxycarbonyl-2-benzyloxy-3-allylguanidine (78). To a solution of 79 (1.50 g, 6.87

mmol), O-benzylhydroxylamine (1.25 mL, 10.3 mmol), and diisopropylethylamine (1.26 mL,

7.56 mmol) in CH2Cl2 (68.7 mL) at 0 C was added EDCI (2.63 g, 13.7 mmol, 2 equiv). The

reaction mixture was stirred for 1 h, warmed to rt, stirred for 14 h, and quenched with 1 M HCl

(5 mL). The organic solution was washed with water (10 mL) and brine (10 mL), dried

(Na2SO4), concentrated, and purified by chromatography on SiO2 (2:1, hexanes/EtOAc to 1:1

hexanes/EtOAc) to give 78 (1.78 g, 76%) as a clear oil: IR (ATR) 3386, 1726, 1640 cm-1

; 1H

NMR (300 MHz, CDCl3) 7.96 (s, 1 H), 7.48–7.29 (m, 10 H), 6.41 (bs, 1 H), 5.99 (dddd, 1 H, J

= 1.2, 5.7, 11.4, 15.9 Hz), 5.20 (m, 2 H), 5.15 (s, 2 H) 4.90 (s, 2 H), 3.79-3.75 (m, 2 H); 13

C

NMR (75 MHz, CDCl3) 153.0, 148.1, 137.7, 135.2, 134.4, 128.9, 128.8, 128.5, 128.4, 128.1,

116.1, 76.0, 67.8, 43.4; HRMS (EI+) m/z calcd for C19H21N3O3

([M]

+.) 339.1583, found

339.1581.

57

Methyl 3,4-(1-benzyloxy-2-benzyloxycarbonyl)guanidinobutanoate (77) and methyl 3,4-(1-

benzyloxycarbonyl-2-benzyloxy)guanidinobutanoate (80). A 50-mL, 3-necked, round-

bottomed flask fitted with rubber septum and a three-way hose adapter (with stopcock, equipped

with a vacuum inlet, and a balloon filled with CO) was charged with Pd(OAc)2 (56.1 mg, 0.250

mmol), CuCl2 (1.02 g, 7.49 mmol), and degassed CH3OH (4.0 mL). The flask was evacuated

and back-filled with CO (3x), cooled to 0 C, and stirred for 0.5 h. To this mixture was added a

solution of 78 (848 mg, 2.50 mmol) in degassed CH3OH (4.32 mL). The reaction mixture was

stirred at 0 C for 18 h, warmed to rt, stirred for 4 h, and concentrated. The residue was

dissolved in EtOAc, and filtered through celite. The brownish filtrate was washed with sat.

NaHCO3 (1 x 5 mL), ethylenediamine (1 x 1 mL), and sat. NaHCO3 (1 x 5 mL). The washing

procedure was repeated until the organic layer remained yellow. The organic solution was dried

(Na2SO4), concentrated, and purified by chromatography on florisil (1:1, hexanes/EtOAc to

100% EtOAc) to give 77 and 80 (750 mg, 76%) in a isomeric ratio of 2.2:1 as a yellow oil that

was not further separated: 1H NMR (300 MHz, CDCl3) 7.43–7.26 (m, 20 H), 5.26 (ad, 2 H, J =

1.5 Hz, minor isomer 77), 5.16 (s, 2 H, major isomer 77), 5.07 (d, 1 H, J = 11.4 Hz, major isomer

77), 5.00 (d, 2 H, J = 11.4 Hz, major isomer 77), 4.98 (s, 2 H, minor isomer 80).

58

Methyl 3,4-(1-benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidinobutanoate

(77-Boc) and methyl 3,4-(1-benzyloxycarbonyl-2-benzyloxy-3-tert-butoxycarbonyl)

guanidinobutanoate (80-Boc). A 50-mL, 3-necked, round-bottomed flask fitted with rubber

septum and a three-way hose adapter (with stopcock, equipped with a vacuum inlet, and a

balloon filled with CO) was charged with Pd(OAc)2 (166 mg, 0.737 mmol), CuCl2 (3.00 g, 22.1

mmol), and degassed CH3OH (12.0 mL). The flask was evacuated and back-filled with CO (3x),

cooled to 0 C, and stirred for 0.5 h. To this mixture was added a solution of 78 (2.50 g, 7.37

mmol) in degassed CH3OH (12.6 mL) and the resulting solution was stirred at 0 C for 18 h,

warmed to rt, stirred for 4 h, and concentrated. The residue was dissolved in EtOAc, filtered

through celite, and the brownish filtrate was washed with sat. NaHCO3 (10 mL),

ethylenediamine (1 mL), and sat. NaHCO3 (10 mL). The washing procedure was repeated until

the organic layer remained yellow. The organic solution was dried (Na2SO4) and concentrated

under reduced pressure. The crude mixture of 77 and 80 was dissolved in THF (73.7 mL) and

treated with Boc2O (1.93 g, 8.84 mmol, 1.2 equiv). To this solution was added triethylamine

(2.30 mL, 1.66 mmol) followed by DMAP (182 mg, 1.47 mmol, 0.2 equiv) and the resulting

mixture was stirred for 24 h, treated with sat. NH4Cl (20 mL), and extracted with EtOAc (3 x 20

mL). The combined organic extracts were dried (Na2SO4), concentrated, and purified by

chromatography on SiO2 (2:1, hexanes/EtOAc) to give 77-Boc (1.40 g, 38%) as a white solid:

59

Mp 123.3–124.5 C;

IR (ATR) 1752, 1724, 1687, 1631 cm-1

; 1H NMR (300 MHz, CDCl3)

7.39–7.25 (m, 10 H), 5.02 (s, 2 H), 4.95 (d, 1 H, J = 10.5 Hz), 4.81 (d, 1 H, J = 10.5 Hz), 3.92–

3.82 (m, 2 H), 3.62 (s, 3 H), 3.47–3.42 (m, 1 H), 2.77 (dd, 1 H, J = 3.9, 16.5 Hz), 2.32 (dd, 1 H, J

= 8.4, 16.5 Hz), 1.48 (s, 9 H); 13

C NMR (75 MHz, CDCl3) 170.8, 160.0, 149.7, 136.4, 135.4,

129.5, 129.0, 128.7, 128.6, 128.4, 128.0, 83.9, 67.9, 52.2, 28.2; HRMS (EI+) m/z calcd for

C26H31N3O7 ([M]+) 497.2162, found 497.2160: and 80-Boc (0.834 g, 23%) as a yellow oil: IR

(ATR) 1733, 1724, 1709, 1653 cm-1

; 1H NMR (300 MHz, CDCl3) 7.48–7.25 (m, 10 H), 5.30

(d, 1 H, J = 12.6 Hz), 5.23 (d, 1 H, J = 12.3 Hz), 5.05 (s, 2 H), 4.49 (ddd, 1 H, J = 3.3, 6.9, 12.9

Hz), 3.78–3.76 (m, 1 H), 3.68 (s, 3), 3.67 (dd, 1 H, J = 3.9, 6.9 Hz), 2.87 (dd, 1 H, J = 3.3, 16.2

Hz), 2.46 (dd, 1 H, J = 9.9, 16.2 Hz); 13

C NMR (75 MHz, CDCl3) 170.6, 151.6, 151.0, 141.9,

137.4, 135.6, 129.9, 128.9, 128.7, 128.5, 128.3, 128.2, 128.0, 82.9, 68.3, 52.7, 52.2, 50.0, 37.2,

28.0; HRMS (ES+) m/z calcd for C26H31N3O7Na ([M + Na]

+) 520.2060, found 520.2038.

3,4-(1-Benzyloxy-2-benzyloxycarbonyl)guanidinobutanoic acid (87). To a solution of 77 and

70 (0.350 g, 0.881 mmol) in Et2O/THF (35.2 mL, 10:1 v/v) was added KOTMS (0.314 g, 2.20

mmol) and the resulting mixture was stirred for 3 h. After the addition of water (10 mL) the

biphasic solution was vigorously stirred for 5 min, the aqueous phase was separated, acidified

(pH = 2) with 1 M HCl, and extracted with EtOAc (5 x 10 mL). The combined organic extracts

60

were dried (Na2SO4) and concentrated to give of 87 (233 mg, 69%) as a powder: Mp 150.1–

151.9 C; IR (ATR) 3340, 1702, 1638, 1610 cm-1

. 1H NMR (300 MHz, CDCl3) 7.43–7.26 (m,

10 H), 5.16 (s, 2 H), 5.05 (d, 1 H, J = 11.4 Hz), 4.98 (d, 1 H, J = 11.1 Hz), 3.86–3.77 (m, 1 H),

3.67 (dd, 1 H, J = 7.8, 9.6 Hz), 3.17 (dd, 1 H, J = 7.2, 9.6 Hz), 2.63 (dd, 1 H, J = 3.9, 16.8 Hz),

2.19 (dd, 1 H, J = 9.3, 16.8 Hz); 13

C NMR (75 MHz, CDCl3) 173.2, 166.7, 164.3, 137.0,

136.0, 130.0, 129.0, 128.7, 128.6, 128.3, 128.1, 78.8, 67.5, 57.3, 45.3, 34.5; HRMS (EI+) m/z

calcd for C20H21N3O5 ([M]+.

) 383.1481, found 383.1479.

Methyl-3,4-(1-benzyloxy-2-benzyloxycarbonyl)guanidinebutanoyloxy(methyl)

carbamodithioate (94). A solution of 87 (0.250 g, 0.652 mmol) in CH2Cl2 (10.0 mL) at 0 C

was treated with DCC (161 mg, 0.717 mmol) and DMAP (8.05 mg, 0.0652 mmol). After 0.5 h

the flask was protected from light and a solution of 9247

(89.5 mg, 0.652 mmol) in CH2Cl2 (3.0

mL) was added slowly. The mixture was warmed to rt, stirred for 24 h, filtered through celite,

concentrated, and purified by chromatography on SiO2 (1:1, hexanes/EtOAc) to give of 94 (248

mg, 76%) as a foam: IR (ATR) 3319, 1621, 1569, 1536 cm-1

; 1H NMR (300 MHz, CDCl3)

8.03 (bs, 1 H), 7.46–7.29 (m, 10 H), 5.17 (s, 2 H), 5.02 (s, 2 H), 3.95–3.86 (m, 1 H), 3.72 (dd, 1

H, J = 8.0, 9.9 Hz), 3.65 (s, 3 H), 3.27 (dd, 1 H, J = 6.3, 9.9 Hz), 2.76 (dd, 1 H, J = 4.5, 16.8 Hz),

61

2.53 (s, 3 H), 2.33 (dd, 1 H, J = 9.0, 16.8 Hz); 13

C NMR (75 MHz, CDCl3) 196.9, 167.4,

166.5, 164.5, 137.0, 136.0, 130.2, 129.2, 128.8, 128.6, 128.4, 128.1, 78.8, 67.5, 56.9, 45.0, 42.8,

32.4, 19.0; MS (ES+) m/z 525 ([M + Na]

+, 70), 503 (100), 247 (80), 212 (85).

Methyl-3,4-(1-benzyloxy-2-benzyloxycarbonyl)guanidinobutanoyloxy(phenyl)

carbamodithioate (95). A solution of 87 (0.250 g, 0.652 mmol) in CH2Cl2 (10.0 mL) at 0 C

was treated with DCC (161 mg, 0.717 mmol) and DMAP (8.05 mg, 0.0652 mmol). After 0.5 h

the flask was protected from light and a solution of 9347

(0.130 g, 0.652 mmol) in CH2Cl2 (3.0

mL) was added slowly. The mixture was warmed to rt, stirred for 24 h, filtered through celite,

concentrated, and purified by chromatography on SiO2 (1:1, hexanes/EtOAc) to give 95 (207 mg,

56%) as a yellow oil: IR (ATR) 3360, 1653, 1603, 1577 cm-1

; 1H NMR (300 MHz, CDCl3)

7.99 (bs, 1 H), 7.48–7.26 (m, 15 H), 5.17 (s, 2 H), 5.03 (d, 1 H, J = 11.4 Hz), 4.98 (d, 1 H, J =

11.4 Hz), 3.89–3.80 (m, 1 H), 3.69 (dd, 1 H, J = 7.8, 9.9 Hz), 3.28 (dd, 1 H, J = 6.6, 9.9 Hz),

2.78 (dd, 1 H, J = 3.9, 16.8 Hz), 2.58 (s, 3 H), 2.32 (dd, 1 H, J = 9.9 Hz, 16.5 Hz); 13

C NMR (75

MHz, CDCl3) 197.6, 167.3, 166.6, 164.5, 137.0, 136.0, 130.0, 129.8, 129.0, 128.7, 128.6,

128.5, 128.4, 128.0, 78.7, 77.4, 67.5, 57.1, 45.0, 32.3, 19.8; HRMS (ES+) m/z calcd for

C28H29N4O5S2 ([M + H]+) 565.1579, found 565.1559.

62

3,4-(1-Benzyloxy-2-benzyloxycarbonyl)guanidinobutanamide (97). A sealed tube was

charged with a solution of 77 and 80 (0.250 g, 0.629 mmol, 1:1) in CH3OH (2.1 mL), cooled to -

78 C, and treated with liquid ammonia (2.1 mL). The tube was sealed, slowly warmed to rt

over 1 h, stirred 13 h, and concentrated to give 97 (153 mg, 64%) as a white solid: Mp 141.8–

143.5 C; IR (ATR) 3362, 3178, 1668, 1649, 1579 cm-1

; 1H NMR (300 MHz, CDCl3) 7.93

(bs, 1 H), 7.44–7.26 (m, 10 H), 5.65 (bs, 1 H), 5.23 (bs, 1 H), 5.18 (dd, 1 H, J = 12.6 Hz), 5.14

(dd, 1 H, J = 12.6 Hz), 5.02 (s, 2 H), 3.90–3.81 (m, 1 H), 3.65 (dd, 1 H, J = 7.8, 9.9 Hz), 3.23

(dd, 1 H, J = 6.0, 9.9 Hz), 2.41 (dd, 1 H, J = 4.2, 15.6 Hz), 2.10 (dd, 1 H, J = 9.0, 15.3 Hz); 13

C

NMR (75 MHz, CDCl3) 171.8, 166.3, 164.3, 137.1, 135.9, 130.0, 129.0, 128.7, 128.6, 128.4,

128.1, 78.5, 67.4, 57.3, 45.0, 35.1; HRMS (ES+) m/z calcd for C20H22N4O4Na ([M + Na]

+)

405.1539, found 405.1525.

63

(Z)-2-(4-(tert-Butyldiphenylsilyloxy)but-2-enyl)phthalimide (100). To a solution of (Z)-(4-

bromobut-2-enyloxy)(tert-butyl)diphenylsilane (13.4 g, 34.5 mmol) in DMF (98.5 mL) was

added potassium phthalimide (9.78 g, 51.7 mmol). The resulting mixture was heated to 80 C

for 25.5 h, cooled to rt, concentrated, and diluted with CH2Cl2 (100 mL). The crude solution was

filtered through SiO2, concentrated, and purified by chromatography on SiO2 (5:1,

hexanes/EtOAc) to give 100 (11.8 g, 75%) as a pale yellow oil: IR (ATR) 3068, 1709 cm-1

; 1H

NMR (300 MHz, CDCl3) 7.84–7.69 (m, 8 H), 7.48–7.37 (m, 6 H), 5.85-5.76 (m, 1 H), 5.54–

5.45 (m, 1 H), 4.49 (td, 2 H, J = 0.6, 6.0 Hz), 4.20 (td, 2 H, J = 0.6, 6.9 Hz), 1.09 (2, 9 H); 13

C

NMR (75 MHz, CDCl3) 168.0, 135.8, 134.1, 133.7, 132.3, 129.9, 127.9, 124.2, 123.4, 60.4,

35.2, 27.0, 19.3; HRMS (EI+) m/z calcd for C27H26NO3Si ([M – 15]

+) 440.1682, found

440.1694.

64

(Z)-4-(3-(Benzyloxycarbonyl)thiourea)but-2-enyl-tert-butyldiphenylsilyloxane (101). To a

solution of 100 (11.3 g, 24.9 mmol) in EtOH (258 mL) was added hydrazine monohydrate (2.54

g, 49.8 mmol). The mixture was heated at reflux for 4 h, cooled to 0 C, filtered, and

concentrated. The crude residue was added at 0 C to a solution of CbzNCS41

(7.21 g, 37.3

mmol) in CH2Cl2 (258 mL). The solution was stirred for 15 h while slowly warming to rt,

washed with 1 M HCl (25 mL), water (25 mL), and brine (25 mL). The organic phase was dried

(Na2SO4) and concentrated. The crude material was purified by chromatography on SiO2 (9:1,

hexanes/EtOAc) to give 101 (12.3 g, 96%) as a yellow oil: IR (ATR) 3248, 3287, 1715, 1526

cm-1

; 1H NMR (300 MHz, CDCl3) 9.54 (bs, 1 H), 8.25 (s, 1 H), 7.72–7.67 (m, 4 H), 7.45–7.37

(m, 11 H), 5.86–5.78 (m, 1 H), 5.56-5.48 (m, 1 H), 5.18 (s, 2 H), 4.32 (dd, 2 H, J = 0.9, 6 Hz),

4.14 (t, 2 H, J = 5.4 Hz), 1.08 (s, 9 H); 13

C NMR (75 MHz, CDCl3) 179.0, 152.6, 135.7, 134.7,

133.6, 133.4, 129.9, 129.2, 128.9, 128.5, 127.9, 124.4, 77.2, 68.3, 60.4, 43.1, 27.0, 19.3; HRMS

(ES+) m/z calcd for C29H34N2O3SiSNa ([M + Na]

+) 541.1957, found 541.1957.

65

(Z)-4-(2-(Benzyloxy)-3-(benzyloxycarbonyl)guanidino)but-2-enyl-tert-butyldiphenylsilylane

(102). To a solution of 101 (1.00 g, 1.99 mmol) and O-benzylhydroxylamine hydrochloride

(0.397 g, 2.39 mmol) in CH2Cl2 (13.3 mL) was added diisopropylethylamine (0.697 mL, 4.18

mmol). The resulting mixture was cooled to 0 C, treated with EDCI (6.41 g, 33.4 mmol, 1.5

equiv), stirred for 1 h, warmed to rt, and stirred for 41 h. The solution was washed with 1 M HCl

(5 mL), water (5 mL), and brine (5 mL). The organic phase was dried (Na2SO4), concentrated,

and purified by chromatography on SiO2 (2:1, hexanes/EtOAc) to give 102 (0.84 g, 71%) as a

yellow oil: IR (ATR) 3390, 1728, 1646 cm-1

; 1H NMR (300 MHz, CDCl3) 7.92 (s, 1 H),

7.76–7.74 (m, 4 H), 7.47–7.32 (m, 16 H), 6.23 (bt, 1 H, J = 5.4 Hz), 5.81–5.75 (m, 1 H), 5.56–

5.50 (m, 1 H), 5.15 (s, 2 H), 4.85 (s, 2 H), 4.37–4.34 (m, 2 H), 3.62 (t, 2 H, J = 5.7 Hz), 1.12 (s, 9

H); 13

C NMR (75 MHz, CDCl3) 152.9, 147.9, 137.7, 135.8, 135.3, 133.8, 132.0, 129.9, 129.4,

128.9 (2), 128.6, 128.5, 128.1, 127.9, 127.8, 126.8, 76.1, 67.8, 60.5, 38.4, 27.0, 19.3; HRMS

(EI+) m/z calcd for C37H41N3O4 ([M]

+.) 591.3097, found 591.3080.

66

(Z)-4-(3-(Benzyloxycarbonyl)thioureido)but-2-enyl benzoate (103). A solution of 102 (8.19

g, 16.3 mmol) in THF (54.3 mL) was treated with TBAF (32.6 mL, 32.6 mmol, 1 M in THF) and

stirred for 24 h. The solution was diluted with water (40 mL), extracted with EtOAc (3 x 40 mL),

dried (Na2SO4), and concentrated. The crude oil was dissolved in pyridine (163 mL) and cooled

to 0 °C. The resulting solution was treated with DMAP (1.01 g, 8.16 mmol) and BzCl (0.508 mL,

4.38 mmol) was added over 5 min via syringe. The solution was warmed to rt, stirred for 12.5 h,

treated with sat. NH4Cl (50 mL), and stirred for 1 h. The mixture was diluted with H2O (25 mL)

and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with 1 M

HCl (3 x 25 mL), dried (Na2SO4), concentrated, and purified by chromatography on SiO2 (4:1,

hexanes/EtOAc) to give 103 as a white solid: Mp 75.2–76.8 °C; IR (ATR) 3287, 1713, 1521

cm-1

; 1H NMR (300 MHz, CDCl3) 9.73 (bs, 1 H), 8.15 (s, 1 H), 8.07–8.04 (m, 2 H), 7.57 (mt,

1 H, J = 7.5 Hz), 7.47–7.36 (m, 7 H), 5.93–7.75 (m, 2 H), 5.17 (s, 2 H), 4.95 (d, 2 H, J = 6.6 Hz),

4.47 (t, 2 H, J = 6.0 Hz); 13

C NMR (75 MHz, CDCl3) 179.4, 166.6, 152.6, 134.7, 133.3, 129.9,

129.1, 129.0, 128.7, 128.6, 128.5, 127.9, 68.4, 60.6, 42.9; HRMS (ES+) m/z calcd for

C20H20N2O4SNa ([M + Na]+) 407.1041, found 407.1021.

67

(Z)-4-(2-(Benzyloxy)-3-(benzyloxycarbonyl)guanidino)but-2-enyl benzoate (99). To a

solution of 103 and O-benzylhydroxylamine hydrochloride (3.15 g, 19.6 mmol, 1.2 equiv) in

CH2Cl2 (81.5 mL) was added diisopropylamine (5.98 mL, 35.8 mmol). The resulting mixture

was cooled to 0 °C, treated with EDCI (6.25 g, 32.6 mmol), stirred for 1 h, warmed to rt, and

stirred for 12 h. The solution was washed with 1 M HCl (80 mL), water (80 mL), and brine (80

mL). The organic phase was dried (Na2SO4), concentrated, and purified by chromatography on

SiO2 (2:1 hexanes/EtOAc) to give 99 (4.93 g, 50%) over two steps as a yellow oil: IR (ATR)

3388, 1726, 1646, 1452 cm-1

; 1H NMR (300 MHz, CDCl3) 8.08–8.03 (m, 2 H), 7.90 (bs, 1 H),

7.56 (tt, 1 H, J = 1.2, 6.6 Hz), 7.46–7.24 (m, 12 H), 6.37 (bt, 1 H, J = 5.4 Hz), 5.84–5.72 (m, 2

H), 5.12 (s, 2 H), 4.93 (d, 2 H, J = 5.4 Hz), 4.87 (s, 2 H), 3.87 (t, 2 H, J = 5.7 Hz); 13

C NMR (75

MHz, CDCl3) 153.0, 147.8, 137.7, 135.2, 131.0, 129.9, 128.9 (2), 128.6, 128.5 (2), 128.1,

126.4, 76.11, 67.9, 60.9, 38.3; HRMS (EI+) m/z calcd for C27H27N3O5 ([M]

+.) 473.1951, found

473.1951.

68

3,4-(1-Benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidinobut-1-ene (104). A

solution of 99 (25.0 mg, 0.0528 mmol) in THF (0.264 mL) was treated with PdCl2(CH3CN)2

(2.77 mg, 0.0106 mmol, 0.2 equiv) and stirred overnight. To the solution was added Boc2O

(14.3 mg, 0.0634 mmol), DMAP (2.61 mg, 0.0211 mmol), and triethylamine (8.24 µL, 0.0581

mmol). The reaction mixture was stirred for 6 h, quenched with sat. NH4Cl (2 mL), extracted

with EtOAc (3 x 2 mL), the combined organic extracts were dried (Na2SO4), and concentrated.

The crude mixture was purified by chromatography on SiO2 (1:1, hexanes/EtOAc) to give 104

(20.0 mg, 84%) as a yellow oil: IR (ATR) 1761, 1692 cm-1

; 1H NMR (300 MHz, CDCl3)

7.38–7.18 (m, 10 H), 5.79 (ddd, 1 H, J = 8.7, 9.9, 17.1 Hz), 5.39 (d, 1 H, J = 17.1 Hz), 5.33 (d, 1

H, J = 10.5 Hz), 5.01 (d, 1 H, J = 9.9 Hz), 5.00 (d, 1 H, J = 12.3 Hz), 4.85 (d, 1 H, J = 11.7 Hz),

4.85 (d, 1 H, J = 10.5 Hz), 4.02 (aq, 1 H, J = 8.4 Hz), 3.83 (dd, 1 H, J = 8.1, 10.2 Hz), 3.39 (dd, 1

H, J = 8.7, 10.5 Hz), 1.49 (s, 9 H). 13

C NMR (75 MHz, CDCl3) 160.3, 149.7, 149.4, 136.3,

135.4, 133.7, 129.4, 128.8, 128.6, 128.5, 128.5, 128.0, 122.2, 83.7, 77.9, 67.9, 63.7, 46.9, 28.2;

HRMS (EI+) m/z calcd for C25H29N3O5 ([M]

+.) 451.2107, found 451.2106.

General procedure for chiral ligand screen in the amidoalkylation of compound 104. A

solution of PdCl2(CH3CN)2 (0.2 equiv) and ligand (0.4 equiv) in THF was stirred for 40 min. To

this mixture was added a solution of 99 in THF and the resulting solution was stirred overnight.

After the addition of THF (to a final concentration of 0.2 M), Boc2O (1.2 equiv), DMAP (0.4

69

equiv), and triethylamine (1.1 equiv), the mixture was stirred for 6–24 h, quenched with sat.

NH4Cl, and extracted with EtOAc (3x). The combined organic extracts were dried (Na2SO4),

concentrated, and purified by chromatography on SiO2 (1:1, hexanes/EtOAc).

2,3-(1-Benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidinopropanol (108).

Ozone was bubbled through a solution of 104 (2.62 g, 5.80 mmol) and pyridine (4.69 mL, 58.0

mmol) in CH3OH/CH2Cl2 (69.6 mL/ 46.4 mL, 0.05 M) at -78 C for 15 min. Argon was bubbled

through the solution for 5 min, NaBH4 (329 mg, 8.70 mmol) was added, the mixture was stirred

for 1 h, and slowly warmed to rt overnight. The solution was quenched with water (25 mL),

extracted with EtOAc (4 x 25 mL), the combined organic extracts were dried (Na2SO4),

concentrated, and purified by chromatography on SiO2 (1:1 hexanes/EtOAc to 100% EtOAc) to

give 108 (1.51 g, 57%) as a white foam: IR (ATR) 3440, 1752, 1690 cm-1

; 1H NMR (300 MHz,

CDCl3) 7.44–7.27 (m, 10 H), 5.09 (s, 2 H), 4.95 (s, 2 H), 3.73–3.59 (m, 2 H), 3.57 (dd, 1 H, J =

3.3, 12.3 Hz), 3.51–3.44 (m, 1 H), 3.40 (dd, 1 H, J = 2.4, 12.3 Hz), 1.98 (bs, 1 H), 1.47 (s, 9 H);

13C NMR (75 MHz, CDCl3) 160.0, 151.2, 149.7, 136.3, 135.7, 129.7, 129.0, 128.7, 128.5,

128.3, 127.9, 83.6, 77.5, 67.9, 59.7, 59.4, 44.2, 28.0; HRMS (EI+) m/z calcd for C24H29N3O6

([M]+.

) 455.2056, found 455.2074.

70

2,3-(1-Benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidin-1-azido-propane

(113). To a solution of 108 (0.500 g, 1.08 mmol), triethylamine (0.467 mL, 3.29 mmol), and

DMAP (27.1 mg, 0.220 mmol) in CH2Cl2 (5.40 mL) at 0 C was added MsCl (0.174 mL, 2.20

mmol). The reaction mixture was stirred for 4.5 h at 0 C, warmed to rt, and stirred for 1.5 h.

The solution was diluted with CH2Cl2 (10 mL), and washed with sat. Na2CO3 (10 mL), brine (10

mL), dried (Na2SO4), and concentrated. The crude residue was dissolved in DMF (5.50 mL),

treated with NaN3 (79.3 mg, 1.23 mmol), and placed in a preheated 65 C oil bath for 6 h. The

solution was cooled to rt, diluted with EtOAc, washed with water, dried (Na2SO4), and

concentrated. The crude material was purified by chromatography on SiO2 (1:1 hexanes/EtOAc)

to give 113 (405 mg, 77%) as a clear oil: IR (ATR) 2102, 1756, 1690 cm-1

; 1H NMR (300

MHz, CDCl3) 7.43–7.26 (m, 10 H), 5.11 (d, 1 H, J = 12.3 Hz), 5.06 (d, 1 H, J = 12.3 Hz), 4.96

(d, 1 H, J = 11.1 Hz), 4.76 (d, 1 H, J = 11.1 Hz), 3.70 (dd, 1 H, J = 8.1, 10.2 Hz), 3.51 (dd, 1 H, J

= 5.4, 10.2 Hz), 3.47–3.40 (m, 1 H), 3.32 (d, 2 H, J = 4.5 Hz), 1.49 (s, 9 H); 13

C NMR (75 MHz,

CDCl3) 159.9, 149.7, 149.6, 136.5, 135.7, 129.6, 129.1, 128.8, 128.6, 128.4, 128.0, 84.0, 78.2,

67.9, 57.3, 50.2, 45.0, 28.1; HRMS (EI+) m/z calcd for C24H28N6O5 ([M]

+) 480.2121, found

480.2132.

71

2,3-(1-Benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidino-1-(N-tert-

butoxycarbonyl)propylamine (115). To a solution of 113 (0.183 mg, 0.381 mmol), Boc2O

(103 mg, 0.457 mmol), and Pd/C (40.5 mg, 0.0381 mmol) in EtOAc (3.81 mL) was added

triethylamine (0.0703 mL, 0.495 mmol). The reaction vessel was evacuated and back-filled with

H2 (3x), stirred for 2 h, and the hydrogen balloon was removed. The reaction mixture was stirred

for an additional 3 h and filtered through celite. The organic solution was washed with water (3

x 5 mL), dried (Na2SO4), concentrated, and purified by chromatography on SiO2 (1:1,

hexanes/EtOAc) to give 113 (0.110 g, 52%): IR (ATR) 3364, 1757, 1703 cm-1

; 1H NMR (300

MHz, CDCl3) 7.45–7.21 (m, 10 H), 5.12 (d, 1 H, J = 12.3 Hz), 5.07 (d, 1 H, J = 12.3 Hz), 4.97

(d, 1 H, J = 11.4 Hz), 4.93 (d, 1 H, J = 11.1 Hz), 4.10 (bs, 1 H), 3.68 (dd, 1 H, J = 8.1, 9.9 Hz),

3.54–3.49 (m, 1 H), 3.46–3.41 (m, 1 H), 3.37 (m, 1 H), 3.00–2.94 (m, 1 H), 1.46 (s, 9 H), 1.41 (s,

9 H); 13

C NMR (75 MHz, CDCl3) 159.9, 156.2, 149.8, 136.5, 135.8, 130.1, 129.2, 128.8,

128.6, 128.4, 128.0, 83.8, 79.9, 68.0, 58.3, 44.7, 39.2, 28.4, 28.1; HRMS (ES+) m/z calcd for

C24H38N4O7Na ([M + Na]+) 577.2638, found 577.2604.

72

2,3-(1-Benzyloxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidino-1-(N-tert-

butoxycarbonyl, N-methyl)propylamine (116). A solution of 115 (50.0 mg, 0.090 mmol) and

CH3I (0.112 mL, 1.80 mmol) in DMF (1.80 mL) at -20 ºC was treated with NaH (3.42 mg, 0.135

mmol). After 30 min, the solution was warmed to 0 ºC, stirred for 30 min, then warmed to rt.

After 30 min, the reaction was quenched with water, extracted with EtOAc, and the combined

organic extracts were dried (Na2SO4) and concentrated. The crude residue was purified by

chromatography on SiO2 (1:1 hexanes/EtOAc) to give 116 (40.4 mg, 79%): IR (ATR) 1759,

1688 cm-1

; 1H NMR (300 MHz, CDCl3, 50 ºC) 7.41–7.26 (m, 10 H), 5.11 (d, 1 H, J = 12.3

Hz), 5.06 (d, 1 H, J = 12.3 Hz), 5.00 (d, 1 H, J = 11.1 Hz), 4.81 (d, 1 H, J = 10.8 Hz), 3.66 (d, 2

H, J = 6.6 Hz), 3.59–3.55 (m, 1 H), 3.35 (d, 2 H, J = 6.3 Hz), 2.75 (s, 3 H), 1.47 (s, 9 H), 1.44 (s,

9 H); 13

C NMR (75 MHz, CDCl3) 159.9, 149.9, 149.6, 136.9, 136.0, 129.7, 129.0, 128.8,

128.6, 128.5, 128.0, 83.8, 78.1, 67.9, 57.7, 48.7, 45.9, 28.6, 28.3; HRMS (ES+) m/z calcd for

C30H40N4O7Na ([M + Na]+) 591.2795, found 591.2757.

73

2,3-(1-Benzyloxy-2-benzyloxycarbonyl)guanidinodimethylpropylamine (117),

5-((Dimethylamino)methyl)-2-iminioimidazolidin-1-olate (9), and 1-(2-iminoimidazolidin-

4-yl)-N,N-dimethylmethanamine (118).17

A solution of 116 (0.100 g, 0.176 mmol) in THF

(1.41 mL) was treated with TFA (0.352 mL) and stirred for 1 h. The reaction mixture was

quenched with NaHCO3 (3 mL), extracted with EtOAc (5 x 3 mL), dried (Na2SO4), and

concentrated. The crude residue was dissolved in distilled EtOAc (1.06 mL), treated with

paraformaldehyde (6.67 mg, 0.211 mmol) and NaBH3CN (24.4 mg, 0.369 mmol). The resulting

mixture was stirred overnight, quenched with NaHCO3 (3 mL), extracted with EtOAc (5 x 3

mL), dried (Na2SO4), and concentrated to give 117 as a white solid: IR (ATR) 3371, 1655, 1610

cm-1

; 1H NMR (300 MHz, CDCl3) 8.07 (bs, 1 H), 7.43 (s, 5 H), 7.37–7.27 (m, 5 H), 5.18 (s, 2

H), 5.10 (d, 1 H, J = 12.0 Hz), 5.05 (d, 1 H, J = 12.0 Hz), 3.92 (t, 1 H, J = 9.0 Hz), 3.61 (q, 1 H,

J = 9.4 Hz), 3.37 (t, 1 H, J = 9.6 Hz), 2.61 (dd, 1 H, J = 9.0, 13.8 Hz), 2.43 (s, 3 H), 2.40 (s, 3 H),

2.17 (ad, 1 H, J = 14.4 Hz); HRMS (ES+) m/z calcd for C21H26N4O3Na ([M + Na]

+) 405.1903,

found 405.1867. A solution of 117 in MeOH (20 mL) was passed through an H-cube, fitted with

a Pd/C cartridge at 60 psi, and at a flow rate of 1 mL/min. The solution was concentrated and the

residue was purified by HPLC chromatography (flow rate: 0.7 mL/min, column 3 micron C18

100 x 4.6 mm, mobile phase 95:5 water (0.1% TFA): MeOH, wavelength 254 nm) to give an

inseparable mixture of 9 (9.71 mg, 35%): 1H NMR (600 MHz, D2O (DSS)) 4.51–4.46 (m, 1

74

H), 3.94 (dd, 1 H, J = 13.8, 15.0 Hz), 3.75 (dd, 1 H, J = 7.2, 14.4 Hz), 3.52–3.46 (m, 2 H), 3.00

(s, 6 H); and 118 (13.9 mg, 56%): 1H NMR (600 MHz, D2O (DSS)) 4.58–4.53 (m, 1 H), 3.98

(dd, 1 H, J = 9.6, 9.6 Hz), 3.52–3.46 (m, 2 H), 3.38 (dd, 1 H, J = 4.8, 13.8 Hz), 2.97 (s, 6 H).

(Z)-4-(3-((Benzyloxy)carbonyl)thioureido)but-2-en-1-yl benzoate (103). Seven 50 mL plastic

tubes were each charged with 101 (6 x 1.00 g, 1.93 mmol and 1 x 0.88 g, 1.67 mmol) in THF (6

x 19.3 mL and 1 x 17.2 mL). Each of these solutions were treated with HF•pyr (6 x 1.45 mL,

9.64 mmol and 1 x 1.25 mL, 8.43 mmol) and stirred for 5 h. The reactions were combined into a

2-L Erlenmeyer flask, quenched with sat NaHCO3 (0.500 L), and extracted with EtOAc (3 x

0.100 L). The organic layer was dried (Na2SO4), concentrated, and the crude yellow solid was

dissolved in freshly distilled pyridine (133 mL), treated with DMAP (654 mg, 5.30 mmol), and

cooled to 0 ºC. To the resulting solution was added BzCl (1.85 mL, 15.9 mmol) over 5 min via

syringe. The solution was warmed to rt and stirred overnight. To the solution was added sat.

NH4Cl (100 mL), the reaction was stirred for 1 h, and extracted with EtOAc (3 x 100 mL). The

combined organic extracts were washed with water (3 x 100 mL), dried (Na2SO4), concentrated,

and purified by chromatography on SiO2 (4:1 hexanes/EtOAc) to give 4.19 g (82%) of 103 as a

white solid: IR (ATR) 3289, 1709, 1515 cm-1

; 1H NMR (300 MHz, Acetone – d6) 9.96 (s, 1

75

H), 9.85 (s, 1 H), 8.06-8.03 (m, 2 H), 7.64 (tt, 1 H, J = 1.5, 6.6 Hz), 7.54-7.49 (m, 2 H), 7.45-

7.35 (m, 5 H), 5.93-5.82 (m, 2 H), 5.21 (s, 2 H), 5.02 (d, 2 H, J = 5.4 Hz), 4.53 (t, 2 H, J = 5.4

Hz); 13

C NMR (75 MHz, Acetone – d6) 180.8, 166.6, 154.1, 136.5, 133.9, 131.2, 130.2, 129.4

(2), 129.2, 128.9, 127.8, 68.2, 61.3, 43.0; HRMS (ES+) m/z calcd for C20H20N2O4SNa ([M +

Na]+) 407.1041, found 407.1021.

(Z)-4-(2-(Benzyloxy)-3-(benzyloxycarbonyl)guanidino)but-2-enyl benzoate (99). A solution

of 103 (1.00 g, 2.60 mmol) and O-benzylhydroxylamine hydrochloride (648 mg, 3.38 mmol, 1.3

equiv) in CH2Cl2 (13.0 mL) was treated with diisopropylethylamine (1.43 mL) and EDCI (648

mg, 3.38 mmol). The reaction was stirred overnight, quenched with water (15.0 mL), extracted

with CH2Cl2 (3 x 15.0 mL), dried (Na2SO4), and concentrated. The crude material was purified

by chromatography on SiO2 (2:1 hexanes/EtOAc) to give 1.23 g (74%) of 99 as a yellow oil. 1H

spectra for 99 matched with previously recorded data; 1H NMR (300 MHz, CDCl3) 8.06–8.04

(m, 2 H), 7.90 (bs, 1 H), 7.54 (tt, 1 H, J = 1.2, 7.2 Hz), 7.43–7.28 (m, 12 H), 6.37 (bt, 1 H, J =

5.2 Hz), 5.82–5.72 (m, 2 H), 5.09 (s, 2 H), 4.92 (d, 2 H, J = 5.6 Hz), 4.86 (s, 2 H), 3.85 (t, 2 H, J

= 5.6 Hz);

76

N-(Methoxymethoxy)phthalimide (120).59

A solution of N-hydroxyphthalimide (75.0 g, 446

mmol) and diisoproylethylamine (81.9 mL, 491 mmol) in DMF (686 mL) was treated with a

freshly prepared solution of MOMCl60

in toluene (195 mL, 535 mmol, 2.75 M). The solution

was stirred for 5 h, diluted with CH2Cl2 (500 mL), and washed with sat. NaHCO3 (5 x 100 mL).

The organic phase was dried (Na2SO4) and concentrated to give 120 (91.4 g, 99.0 %) as a white

solid. The spectra matched those previously reported: 1H NMR (400 MHz, CDCl3) 7.87 (s, 4

H), 5.09 (s, 2 H), 3.58 (s, 3 H): 13

C NMR (100 MHz, CDCl3) 163.9, 134.7, 129.3, 123.7,

101.6, 58.0; HRMS (ES+) m/z calcd for C10H10NO4 ([M + H]

+) 208.0604, found 208.0603.

(Z)-4-(2-(Methoxymethoxy)-3-(benzyloxycarbonyl)guanidino)but-2-enyl benzoate (121). A

solution of 120 (943 mg, 4.55 mmol) in CH2Cl2 (4.55 mL) and CH3OH (0.592 mL) was treated

with hydrazine monohydrate (223 L, 4.55 mmol). After 5.5 h the resulting precipitate was

filtered and the filtrate was washed with 5 N aq. ammonia (3 x 5 mL). The aqueous layer was

77

extracted with CH2Cl2 (3 x 5 mL), dried (Na2SO4), and concentrated. The resulting crude oil

was dissolved in CH2Cl2 (3.50 mL), treated with a solution of 103 (0.500 g, 1.30 mmol),

diisopropylethylamine (0.543 mL, 3.25 mmol), and EDCI (534 mg, 2.73 mmol) in CH2Cl2 (3.00

mL). The solution was stirred overnight, quenched with water (10 mL), extracted with EtOAc (3

x 10 mL), dried (Na2SO4), and purified by chromatography on SiO2 (2:1 hexanes/EtOAc) to give

121 (337 mg, 61%) as a pale yellow oil: IR (ATR) 3385, 1715, 1646 cm-1

; 1H NMR (300 MHz,

Acetone – d6) 8.49 (s, 1 H), 8.04 (dd, 2 H, J = 1.2, 8.4 Hz), 7.65–7.60 (m, 1 H), 7.52–7.49 (m,

2 H), 7.43–7.33 (m, 5 H), 6.59 (bt, 1 H, J = 5.2 Hz), 5.86–5.76 (m, 2 H), 5.17 (s, 2 H), 5.00 (d, 2

H, J = 5.6 Hz), 4.87 (s, 2 H), 3.92 (t, 2 H, J = 6.0 Hz), 3.36 (s, 3 H); 13

C NMR (100 MHz,

Acetone – d6) 166.5, 153.8, 148.6, 136.7, 133.8, 132.1, 131.2, 130.1, 129.3 (2), 129.1, 129.0,

126.6, 98.7, 68.0, 56.6, 38.6; HRMS (ES+) m/z calcd for C22H25N3O5Na ([M + Na]

+) 450.1641,

found 450.1656.

3,4-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)-guanidinobut-1-ene

(122). A solution of 121 (325 mg, 0.760 mmol) in THF (3.80 mL) was treated with

PdCl2(CH3CN)2 (19.7 mg, 0.0760 mmol) and stirred overnight. To the solution was added

Boc2O (205 mg, 0.912 mmol), DMAP (37.5 mg, 0.304 mmol), and triethylamine (0.162 mL,

1.14 mmol). The reaction was stirred for 4 h, quenched with sat. NaHCO3 (5 mL), extracted with

EtOAc (3 x 5 mL), dried (Na2SO4), and purified by chromatography on SiO2 (2:1

78

hexanes/EtOAc) to give 122 (308 mg, 95.0%) as a yellow oil: IR (ATR) 1761, 1696 cm-1

; 1H

NMR (300 MHz, Acetone – d6) 7.44–7.30 (m, 5 H), 5.93 (ddd, 1 H, J = 8.4, 10.2, 17.1 Hz),

5.44 (d, 4 H, J = 17.1 Hz), 5.32 (d, 1 H, J = 10.5 Hz), 5.13 (d, 1 H, J = 12.3 Hz), 5.07 (d, 1 H, J =

12.6 Hz), 4.73 (d, 1 H, J = 7.5 Hz), 4.61 (d, 1 H, J = 7.5 Hz), 4.19 (q, 1 H, J = 8.1 Hz), 3.94 (dd,

1 H, J = 8.4, 10.2 Hz), 3.49 (dd, 1 H, J = 8.1, 10.2 Hz), 3.30 (s, 3 H), 1.48 (s, 9 H); 13

C NMR

(100 MHz, Acetone – d6) 159.8, 150.3, 150.2, 137.9, 135.4, 129.0, 128.9, 128.5, 120.5, 101.7,

83.3, 67.5, 63.7, 56.7, 47.9, 28.0; HRMS (ES+) m/z calcd for C20H27N3O6Na ([M + Na]

+)

428.1798, found 428.1802. Retention time of enantiomers was determined by chiral SFC

analysis (chiralpak-IC: 250 × 4.5 mm; 2.0 mL/min; 20% methanol; 220 nm detection; Rt1 6.52

min, Rt2 8.39 min).

3,4-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)-guanidinobut-1-ene

(S-122). A solution of 121 (0.500 g, 0.702 mmol) in CH2Cl2 (1.94 mL) was treated with (S)-(+)-

COP-Cl (42.8 mg, 0.0292 mmol) and stirred 3 h. To the solution was added THF (3.91 mL),

Boc2O (0.309 mg, 1.40 mmol), DMAP (57.8 mg, 0.468 mmol), and triethylamine (0.249 mL,

1.75 mmol). The reaction was stirred overnight, quenched with sat. NaHCO3 (6 mL), extracted

with EtOAc (3 x 6 mL), dried (Na2SO4), and concentrated. The crude material was purified by

chromatography on SiO2 (2:1 hexanes/EtOAc) to give 359 mg (76%) of S-122 as a yellow oil:

72.5:27.5 er was determined by chiral SFC analysis (chiralpak-IC: 250 × 4.5 mm; 2.0 mL/min;

79

20% methanol; 220 nm detection; Rt1 6.49 min, Rt2 8.45 min); [α]20

D +14.7 (c 1.00, CHCl3); IR

(ATR) 1761, 1694 cm-1

; 1H NMR (400 MHz, Acetone – d6) 7.43–7.29 (m, 5 H), 5.94 (ddd, 1

H, J = 8.4, 10.4, 16.8 Hz), 5.45 (d, 1 H, J = 17.2 Hz), 5.33 (d, 1 H, J = 10.8 Hz), 5.11 (d, 1 H, J =

12.4 Hz), 5.06 (d, 1 H, J = 12.8 Hz), 4.73 (d, 1 H, J = 7.2 Hz), 4.61 (d, 1 H, J = 7.6 Hz), 4.22 (q,

1 H, J = 8.0 Hz), 3.96 (dd, 1 H, J = 8.0, 10.0 Hz), 3.51 (dd, 1 H, J = 8.0, 10.4 Hz), 3.31 (s, 3 H),

1.49 (s, 9 H); 13

C NMR (100 MHz, Acetone – d6) 159.9, 150.5, 150.4, 138.1, 135.7, 129.1,

129.0, 128.6, 120.5, 101.9, 83.5, 67.7, 63.9, 56.8, 48.0, 28.1; HRMS (ES+) m/z calcd for

C20H27N3O6Na ([M + Na]+) 428.1798, found 428.1762.

3,4-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)-guanidinobut-1-ene

(R-122). A solution of 121 (0.500 g, 1.17 mmol) in CH2Cl2 (1.94 mL) was treated with (R)-(-)-

COP-Cl (42.8 mg, 0.0292 mmol) and stirred 3 h. To the solution was added THF (3.91 mL),

Boc2O (0.309 mg, 1.40 mmol), DMAP (57.7 mg, 0.468 mmol), and triethylamine (0.166 mL,

1.17 mmol). The reaction was stirred overnight, quenched with sat. NaHCO3 (6 mL), extracted

with EtOAc (3 x 6 mL), dried (Na2SO4), and concentrated. The crude material was purified by

chromatography on SiO2 (2:1 hexanes/EtOAc) to give 351 mg (74%) of R-122 as a yellow oil:

27.0:73.0 er was determined by chiral SFC analysis (chiralpak-IC: 250 × 4.5 mm; 2.0 mL/min;

20% methanol; 220 nm detection; Rt1 6.49 min, Rt2 8.38 min); [α]20

D -15.1 (c 1.00, CHCl3)

(46% ee); IR (ATR) 1761, 1694 cm-1

; 1H NMR (400 MHz, Acetone – d6) 7.43–7.23 (m, 5 H),

80

5.94 (ddd, 1 H, J = 8.0, 10.0, 16.8 Hz), 5.45 (d, 1 H, J = 17.0 Hz), 5.33 (d, 1 H, J = 9.8 Hz), 5.11

(d, 1 H, J = 12.4 Hz), 5.06 (d, 1 H, J = 12.8 Hz), 4.73 (d, 1 H, J = 7.6 Hz), 4.61 (d, 1 H, J = 7.6

Hz), 4.23 (q, 1 H, J = 8.0 Hz), 3.96 (dd, 1 H, J = 8.4, 10.4 Hz), 3.51 (dd, 1 H, J = 8.0, 10.4 Hz),

3.31 (s, 3 H), 1.49 (s, 9 H); 13

C NMR (100 MHz, Acetone – d6) 159.9, 150.5, 150.4, 138.1,

135.7, 129.1, 129.0, 128.6, 120.5, 101.9, 83.5, 67.7, 63.9, 56.8, 48.0, 28.1; HRMS (ES+) m/z

calcd for C20H27N3O6Na ([M + Na]+) 428.1798, found 428.1768.

2,3-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidinopropanol

(129). Ozone was bubbled for 5 min through a solution of 122 (1.00 g, 2.47 mmol) in

CH2Cl2/CH3OH 9.87 mL/ 14.8 mL) and pyridine (1.99 mL, 24.7 mmol) at -78 oC. Argon was

bubbled through the solution for 5 min, NaBH4 (0.111 g, 2.96 mmol) was added, the solution

was warmed to 0 oC, and stirred for 4 h. The reaction was quenched with water (25 mL),

extracted with CH2Cl2 (3 x 30 mL), dried (Na2SO4), and purified by chromatography on SiO2

(1:1 hexanes/EtOAc to 100% EtOAc) to give 129 (0.570 g, 1.39 mmol) as a yellow oil: IR

(ATR) 1758, 1696 cm-1

; 1H NMR (300 MHz, Acetone – d6) 7.44–7.30 (m, 5 H), 5.11 (d, 1 H,

J = 12.6 Hz), 5.06 (d, 1 H, J = 12.6 Hz), 4.78 (d, 1 H, J = 7.5 Hz), 4.70 (d, 1 H, J = 7.8 Hz),

3.94–3.73 (m, 4 H), 3.42 (s, 3 H), 1.49 (s, 9 H); 13

C NMR (100 MHz, Acetone – d6) 160.0,

151.3, 150.4, 137.9, 129.0, 128.8, 128.5, 102.1, 83.3, 67.6, 61.0, 60.1, 57.1, 45.5, 28.1; HRMS

(ES+) m/z calcd for C19H27N3O7Na ([M + Na]

+) 432.1747, found 432.1740.

81

2,3-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidin-1-azido-

propane (130). A solution of 129 (0.250 g, 0.611 mmol) and triethylamine (0.260 mL, 1.83

mmol) in CH2Cl2 (3.05 mL) at 0 oC was treated with MsCl (0.0351 mL, 0.444 mmol) followed

by DMAP (9.13 mg, 0.0740 mmol). After 1 h the reaction was quenched with NaHCO3 (3 mL),

extracted with CH2Cl2 (3 x 3 mL), dried (Na2SO4), and concentrated. The residue was dissolved

in DMF (3.05 mL), treated with 3 Å molecular sieves, triethylamine (0.260 mL, 1.83 mmol), and

NaN3 (60.1 mg, 0.916 mmol). The reaction was placed in a preheated 50 oC oil bath, stirred

overnight, diluted with EtOAc (3 mL), washed with water (3 x 3 mL), dried (Na2SO4), and

purified by chromatography on SiO2 (1:1 hexanes/EtOAc) to give 130 (0.230 g, 86.7%) as a

clear oil: IR (ATR) 2104, 1756, 1689 cm-1

; 1H NMR (400 MHz, Acetone – d6) 7.44–7.42 (m,

2 H), 7.39–7.29 (m, 3 H), 5.12 (d, 1 H, J = 12.8 Hz), 5.08 (d, 1 H, J = 12.8 Hz), 4.77 (d, 1 H, J =

7.6 Hz), 4.66 (d, 1 H, J = 7.6 Hz), 3.97–3.90 (m, 3 H), 3.69 (q, 1 H, J = 10.4 Hz), 3.65–3.62 (m,

1 H), 3.43 (s, 3 H), 1.49 (s, 9 H); 13

C NMR (100 MHz, Acetone – d6) 159.9, 150.7, 150.3,

138.2, 129.1, 129.0, 128.6, 102.5, 83.5, 67.6, 59.3, 57.0, 50.6, 45.7, 28.1; HRMS (ES+) m/z

calcd for C19H26N6O6Na ([M + Na]+) 457.1812, found 457.1803.

82

2,3-(1-Methoxymethoxy-2-benzyloxycarbonyl-3-tert-butoxycarbonyl)guanidin-1-(N-tert-

butoxycarbonyl)propylamine (131). A round-bottomed flask charged with 130 (0.500 g, 1.15

mmol), Lindlar's catalyst (245 mg, 0.115 mmol), Boc2O (381 mg, 1.73 mmol), and EtOAc (11.5

mL) was evacuated and backfilled with H2 (3x). After 24 h the H2 atmosphere was removed, the

vessel was placed under nitrogen atmosphere, and stirred overnight. The reaction solution was

filtered through celite and purified by chromatography on SiO2 (2:1 hexanes/EtOAc) to give 468

mg (80.0%) of 131 as an oil: IR (ATR) 3377, 1760, 1700 cm-1

; 1H NMR (600 MHz, Acetone –

d6) 7.43 (d, 2 H, J = 7.2 Hz), 7.37 (t, 3 H, J = 7.2 Hz), 7.31 (m, 1 H), 5.11 (d, 1 H, J = 12.0

Hz), 5.07 (d, 1 H, J = 12.6 Hz), 4.82 (d, 1 H, J = 7.8 Hz), 4.65 (d, 1 H, J = 7.8 Hz), 3.89 (dd, 1 H,

J = 9.0, 9.6 Hz), 3.83–3.79 (m, 1 H), 3.69 (ddd, 1 H, J = 2.4, 7.8, 14.4 Hz), 3.65 (dd, 1 H, J =

7.2, 9.6 Hz), 3.45 (s, 3 H), 3.25 (tt, 1 H, J = 5.4, 5.4, 14.4 Hz), 1.48 (s, 9 H), 1.41 (s, 9 H); 13

C

NMR (100 MHz, Acetone – d6) 159.9, 157.1, 150.9, 150.5, 138.1, 129.1, 129.0, 128.6, 102.3,

83.4, 79.3, 67.6, 60.1, 57.2, 46.0, 40.0, 28.5, 28.1; HRMS (ES+) m/z calcd for C24H36N4O8Na

([M + Na]+) 531.2431, found 531.2418.

83

2,3-(1-Methoxymethoxy-2-benzyloxycarbonyl)guanidin-1-(N-methyl)propylamine (132). A

solution of 131 (0.500 g, 0.983 mmol) and CH3I (1.24 mL, 19.7 mmol) in DMF (98.3 mL) at 0

oC was treated with NaH (24.8 mg, 0.983 mmol) and stirred for 1 h. The reaction was warmed to

rt, stirred for 3 h, quenched with sat. NaHCO3 (10 mL), extracted with EtOAc (3 x 10 mL), dried

(Na2SO4), and concentrated. The crude material was purified by chromatography on SiO2 (1:1

hexanes/EtOAc), dissolved in CH2Cl2 (7.86 mL), treated with TFA (1.97 mL) and stirred for 1 h.

The solution was quenched with NaHCO3 (25 mL), extracted with EtOAc (3 x 25 mL), dried

(Na2SO4), and purified by chromatography on SiO2 (95:5 CH2Cl2/MeOH with 1% Et3N) to give

0.230 g (73%) of 132 as a white solid: IR (ATR) 3485, 3372, 1650, 1599 cm-1

; 1H NMR (400

MHz, CD3CN) 7.37–7.27 (m, 5 H), 5.05 (s, 2 H), 4.98 (d, 1 H, J = 7.6 Hz), 4.80 (d, 1 H, J =

7.6 Hz), 3.79–3.72 (m, 1 H), 3.57 (dd, 1 H, J = 8.8, 9.6 Hz), 3.48 (s, 3 H), 3.44 (dd, 1 H, J = 9.2,

9.6 Hz), 2.84 (dd, 1 H, J = 5.6, 12.4 Hz), 2.79 (dd, 1 H, J = 3.6, 12.8 Hz), 2.38 (s, 3 H); 13

C

NMR (100 MHz, CD3CN) 168.1, 164.7, 138.7, 129.4, 128.8, 128.7, 102.0, 67.3, 62.0, 57.6,

51.2, 44.2, 36.8; HRMS (ES+) m/z calcd for C15H23N4O4 ([M + H]

+) 323.1719, found 323.1714.

84

2,3-(1-Methoxymethoxy-2-benzyloxycarbonyl)guanidin-1-(N-dimethyl)propylamine (133).

A solution of 132 in freshly distilled EtOAc (14.0 mL) was treated with paraformaldehyde (44.1

mg, 1.40 mmol). After 30 min NaCNBH3 (0.111 mg, 1.68 mmol) was added and the reaction

was stirred overnight. The reaction was quenched with water (15 mL), extracted with EtOAc (3

x 15 mL), dried (Na2SO4) and purified by chromatography on SiO2 (97:3 CH2Cl2/MeOH with

1% Et3N) to give 310 mg (66.1%) of 133 as an oil: IR (ATR) 3371, 1650, 1601 cm-1

; 1H NMR

(300 MHz, Acetone – d6) 8.23 (s, 1 H), 7.40–7.28 (m, 5 H), 5.08 (s, 2 H), 4.98 (d, 1 H, J = 7.5

Hz), 4.88 (d, 1 H, J = 7.5 Hz), 3.86–3.71 (m, 2 H), 3.52 (s, 3 H), 3.37 (dd, 1 H, J = 8.4, 8.7 Hz),

2.73 (dd, 1 H, J = 3.9, 12.3 Hz), 2.52 (dd, 1 H, J = 8.7, 12.3 Hz), 2.24 (s, 6 H); 13

C NMR (100

MHz, Acetone – d6) 168.1, 164.6, 138.7, 129.1, 128.6, 128.4, 101.7, 66.9, 60.9, 60.6, 57.1,

46.4, 45.8; HRMS (ES+) m/z calcd for C16H24N4O4 ([M + Na]

+) 359.1695, found 359.1717.

85

2,3-(1-Hydroxy-2-benzyloxycarbonyl)guanidin-1-(N-dimethyl)propylamine (134). A

solution of 133 in CH3OH (0.297 mL, 0.1 M) was treated with a solution of HCl in 1,4-dioxane

(0.0743 mL, 0.149 mmol, 4M solution in 1,4-dioxane), placed in a preheated 50 oC oil bath, and

stirred for 48 h. The solution was concentrated and purified by chromatography on C18 (100%

water) to give 6.4 mg (74%) of 134 as a white solid. Diagnostic peaks for 134: IR (ATR) 3431-

2891, 1745, 1646 cm-1

; 1H NMR (600 MHz, D2O) 7.35–7.31 (m, 5 H), 5.21 (s, 2 H), 4.54 (m,

1 H), 4.03 (dd, 1 H, J = 6.4, 7.2 Hz), 3.69 (dd, 1 H, J = 4.8, 9.6 Hz), 3.60–3.57 (m, 1 H), 3.40

(dd, 1 H, J = 3.2, 9.2 Hz), 2.24 (s, 6 H); 13

C NMR (125 MHz, MeOD) 160.1, 153.1, 135.9,

129.9, 129.8, 129.7, 70.5, 59.8, 58.7; HRMS (ES+) m/z calcd for C14H21N4O3 ([M + H]

+)

293.1614, found 293.1620.

2-(Methylthio)-4,5-dihydro-1H-imidazole (136).61

A solution of ethylenethiourea (1.00 g, 9.79

mmol), CH3OH (1.19 mL), and HI (2.66 mL, 14.7 mmol, 47% wt solution) was placed in a

preheated 75 oC oil bath and stirred overnight. The solution was cooled to rt, treated with 40%

86

NaOH solution until a pH of 11 was reached, and extracted with Et2O (3 x 5 mL). The organic

solution was dried (MgSO4), and concentrated to give 874 mg (77%) of 136 as a solid. The

spectra matched those previously reported: 1H NMR (300 MHz, CDCl3) 5.18 (s, 1 H), 3.42 (s,

4 H), 2.23 (s, 3 H).

2-(Methylthio)-3-benzyloxycarbonyl-4,5-dihydroimidazole (137). A solution of 136 (0.200 g,

1.72 mmol) and triethylamine (0.367 mL, 2.58 mmol) in THF (4.30 mL) was treated with CbzCl

(0.294 mL, 2.07 mmol) and stirred overnight. The reaction mixture was quenched with sat.

NH4Cl (5 mL), extracted with EtOAc, dried (MgSO4), and purified by chromatography on SiO2

(100% CH2Cl2 to 90:10 CH2Cl2/MeOH) to give 427 mg (99%) of 137 as a white solid: Mp 64.7-

66.2 oC; IR (ATR) 1709, 1579 cm

-1;

1H NMR (300 MHz, CDCl3) 7.36–7.32 (m, 5 H), 5.18 (s,

2 H), 3.86 (s, 3 H); 13

C NMR (100 MHz, CDCl3) 158.9, 151.2, 135.1, 128.2, 128.0, 127.8,

67.3, 53.4, 47.0, 14.6; HRMS (ES+) m/z calcd for C12H15N2O2S ([M + H]

+) 251.0854, found

251.0846.

87

2-(Methylthio)-3-benzyloxycarbonyl-4,5-dihydroimidazole (138). A solution of 120 (1.14 g,

5.49 mmol) in CH3CN (11.0 mL) was treated with hydrazine monohydrate (0.523 mL, 5.49

mmol). After 2 h the precipitate was filtered, the filtrate was treated with 137 (275 mg, 5.49

mmol), and Et3N (0.199 mL, 0.137 mmol). The solution was cooled 0 oC, treated with AgNO3

(236 mg, 1.37 mmol), and stirred overnight. The solution was filtered through celite,

concentrated, and purified by chromatography on SiO2 (95% CH2Cl2 5% MeOH) to give 225 mg

(74%) of 138 as a white solid: 1H NMR (300 MHz, CDCl3) 7.43–7.40 (m, 2 H), 7.35–7.28 (m,

3 H), 5.21 (s, 2 H), 4.99 (s, 2 H), 3.87 (t, 2 H, J = 7.2 Hz), 3.43–3.38 (m, 5 H); 13

C NMR (75

MHz, CDCl3) 152.6, 150.7, 135.5, 128.1, 127.8, 127.7, 98.4, 67.4, 56.7, 45.1, 39.8.

2-(N-Hydroxy)-3-benzyloxycarbonyl-4,5-dihydroguanidine (139). A solution of 138 (10.0

mg, 0.0358 mmol) in CH2Cl2 (0.179 mL) at 0 oC was treated with TMSBr (0.0241 mL, 0.179

mmol). After 4 h the solution was quenched with water (1 mL), extracted with CH2Cl2 (3 x 1

mL), and the aqueous layer was concentrated to give 3.42 mg (99%) of 139 as a white solid: 1H

88

NMR (300 MHz, CD3OD) 7.43–7.36 (m, 5 H), 5.31 (s, 2 H), 4.17 (dd, 2 H, J = 8.1, 10.5 Hz),

3.77 (dd, 2 H, J = 6.9, 9.3 Hz).

2-(N-Dimethylphosphate)-3-benzyloxycarbonyl-4,5-dihydroguanidine (140). A solution of

139 (50.0 mg, 0.213 mmol) in CH3CN (1.07 mL) was treated with dimethyl chlorophosphate

(0.0477 mL, 0.425 mmol) and Et3N (0.0302 mL, 2.12 mmol). After 3 days, the reaction was

purified by column chromatography on SiO2 (9:1 ethyl acetate/acetone to 1:1 ethyl

acetate/acetone) to give 22.5 mg (31%) of 140 as a clear oil: IR (ATR) 3336, 1743, 1702 cm-1

;

1H NMR (400 MHz, CD3CN) 7.46–7.33 (m, 5 H), 5.93 (s, 1 H), 5.18 (s, 2 H), 3.90 (dd, 2 H, J

= 7.6, 88 Hz), 3.74 (s, 3 H), 3.71 (s, 3 H), 3.44–3.40 (m, 2 H); 31

P NMR (162 MHz, CD3CN)

1.37; HRMS (ES+) m/z calcd for C13H19N3O6P ([M + H]

+) 344.1011, found 344.0994.

A solution of dimethyl phosphate (0.0756 mL, 0.777 mmol) and triethylamine (0.166 mL, 1.17

mmol) in CH3CN (3.12 mL) at 0 oC was treated with TFAA (0.119 mL, 0.855 mmol). The

solution was warmed to rt, stirred for 10 min, at rt, cooled to 0 oC, treated with a solution of 1-

methylimidazole (70.9 mg, 0.855 mmol) in CH3CN (0.775 mL), and stirred overnight. A portion

of the above solution (3.32 mL, 0.662 mmol, 0.2 M) was added to a solution of 139 (130 mg,

0.553 mmol) in CH3CN (0.360 mL) at 0 oC. The resulting solution was treated with

triethylamine (0.0932 mL, 0.663 mmol), stirred overnight, and purified by column

89

chromatography on SiO2 (9:1 ethyl acetate/acetone to 1:1 ethyl acetate/acetone) to give 93.6 mg

(49.3 %) of 140 as an oil: 1H NMR (400 MHz, CD3CN) 7.48–7.36 (m, 5 H), 6.12 (s, 1 H),

5.21 (s, 2 H), 3.94 (t, 2 H, J = 7.6 Hz), 3.79 (s, 3 H), 3.77 (s, 3 H), 3.46 (t, 2 H, J = 7.6 Hz); 31

P

NMR (162 MHz, CD3CN) 1.71.

2-(N-Diphenylphosphate)-3-benzyloxycarbonyl-4,5-dihydroguanidine (141). A solution of

139 (50.0 mg, 0.213 mmol) in CH3CN (1.06 mL) was treated with diphenyl chlorophosphate

(0.223 mL, 1.06 mmol) and triethylamine (0.0302 mL, 0.213 mmol). After 3 h the resulting

solution was purified by column chromatography on SiO2 (7:3 EtOAc / hexanes) to give 39.6 mg

(40%) of 141 as a yellow oil: 1H NMR (400 MHz, CD3CN) 7.47–7.18 (m, 15 H), 6.02 (s, 1

H), 5.22 (s, 2 H), 3.91 (dd, 2 H, J = 7.6, 8.8 Hz), 3.40 (dd, 2 H, J = 7.6, 8.8 Hz); 31

P NMR (162

MHz, CD3CN) -10.4.

90

3.0 REFERENCES

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94

APPENDIX A

SELECTED 1H AND

13C SPECTRA

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122